WO2023215828A1

WO2023215828A1 - Nanoparticles for inducing a th1 t cell immune response

Info

Publication number: WO2023215828A1
Application number: PCT/US2023/066605
Authority: WO
Inventors: Seth Lederman; Bruce DAUGHERTY
Original assignee: Tonix Pharmacueticlals Holding Corp
Current assignee: Tonix Pharmacueticlals Holding Corp
Priority date: 2022-05-04
Filing date: 2023-05-04
Publication date: 2023-11-09
Anticipated expiration: 2024-11-04

Abstract

Lipid nanoparticles comprising a fusion polynucleotide for use in treating or preventing SARS-CoV-2 infection through adaptive immunity and in particular through cellular CD8 cytotoxic T cell immunity. The fusion polynucleotide comprise different combinations of one or more of a mRNA encoding a SARS-CoV-2 spike protein, a mRNA encoding one or more regions of a surfactant-associated protein D, a mRNA encoding a region of a soluble CD40 ligand, a mRNA encoding a 2A self-cleaving peptide, a mRNA encoding a cell-bound CD40 ligand, a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, and a mRNA encoding a region of a Tissue-type Plasminogen Activator protein.

Description

NANOPARTICLES FOR INDUCING A TH1 T CELL IMMUNE RESPONSE

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority and benefit from United States Provisional Application No. 63/338,217, filed May 4, 2022, the contents of which are hereby incorporated by reference in their entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 4, 2023, is named 104545-0102-W01-SL.xml and is 325,488 bytes in size.

BACKGROUND

[0003] The novel coronavirus is referred to as SARS-CoV-2 or 2019-nCoV and is related to the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), although with only approximately 80% similarity at the nucleotide level. Ralph et al. J Infect Dev Ctries. 2020 Jan 31 ; 14(1):3-17. The infectious disease caused by SARS-CoV-2 is named COVID-19 and its symptoms, depending on the infective strain, include, among others, fever, cough, fatigue, shortness of breath, and loss of smell and taste.

[0004] Coronaviruses are enveloped single stranded RNA viruses with positive-sense RNA genomes ranging from 25.5 to ~32 kb in length. The spherical virus particles range from 70- 120 nm in diameter and contain four structural proteins: the E and M proteins, which form the viral envelope; the N protein, which binds to the virus’s RNA genome; and the S (or Spike) protein, which binds to human receptors. The genome of SARS-CoV-2 also comprises a number of open reading frames that code for a total of nine accessory proteins, which appear to be not essential for virus replication.

[0005] RNA is a polyanionic macromolecule that is susceptible to degradation in biological fluids and cannot readily cross cell membranes to access cellular machinery needed to mediate protein expression. Despite the fact that much effort is currently being invested into RNA-based vaccines to deliver RNA encoding a SARS-CoV-2 S protein and fragments thereof into cells in subjects treated with the RNA, improved vaccines that are able to stimulate adaptive immune responses against viral infection, such as SARS-CoV-2 infection, including, in particular, the activation of Thl T cells and CD8 cytotoxic T cells and, thus, a cellular immune response, are needed.

SUMMARY OF THE DISCLOSURE

[0006] Some embodiments of this disclosure are:

1. A fusion polynucleotide comprising: a. a mRNA encoding a SARS-CoV-2 spike protein or a region thereof; b. a mRNA encoding one or more regions of a surfactant-associated protein D; and c. a mRNA encoding a region of a soluble CD40 ligand.

2. The fusion polynucleotide according to embodiment 1, wherein the fusion polynucleotide further comprises a mRNA encoding a 2A self-cleaving peptide.

3. A fusion polynucleotide comprising: a. a mRNA encoding a region of a SARS-CoV-2 spike protein; and b. a mRNA encoding a region of a soluble CD40 ligand.

4. A fusion polynucleotide comprising: a. a mRNA encoding a SARS-CoV-2 spike protein or a region thereof; b. a mRNA encoding a cell-bound CD40 ligand; and c. a mRNA encoding a 2A self-cleaving peptide.

5. The fusion polynucleotide according to embodiment 4, wherein the cell-bound CD40 ligand is characterized by the amino acid sequence of the full-length protein.

6. The fusion polynucleotide according to embodiment 4, wherein the mRNA encoding a cell-bound CD40 ligand has a deletion of one or more nucleotides.

7. The fusion polynucleotide according to embodiment 5, wherein the cell-bound CD40 ligand has a deletion in the amino acid sequence of the full-length protein extending from amino acid residue 110 to amino acid residue 122.

8. A fusion polynucleotide comprising: a. a mRNA encoding a SARS-CoV-2 spike protein or a region thereof; b. a mRNA encoding an Interferon-a protein; and c. a mRNA encoding a 2A self-cleaving peptide.

9. A fusion polynucleotide comprising: a. a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase; b. a mRNA encoding one or more regions of a surfactant-associated protein D; and c. a mRNA encoding a region of a soluble CD40 ligand.

10. A fusion polynucleotide comprising: a. a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase; and b. a mRNA encoding a region of a Tissue-type Plasminogen Activator protein.

11. The fusion polynucleotide according to embodiment 10, wherein the fusion polynucleotide further comprises a mRNA encoding an Interferon-a protein.

12. The fusion polynucleotide according to embodiment 10, wherein the fusion polynucleotide further comprises a mRNA encoding a region of a soluble CD40 ligand.

13. The fusion polynucleotide according to embodiment 10 or 12, wherein the fusion polynucleotide further comprises a mRNA encoding one or more regions of a surfactant- associated protein D.

14. The fusion polynucleotide according to any one of embodiments 11 or 13, wherein the fusion polynucleotide further comprises a mRNA encoding a 2A self-cleaving peptide.

15. The fusion polynucleotide according to any one of embodiments 1, 9, 13 and 14, wherein one of the one or more regions of the surfactant-associated protein D is an N-terminal region of that protein.

16. The fusion polynucleotide according to embodiment 1, 9, 13 and 14, wherein one of one or more regions of the surfactant-associated protein D is 50 amino acids to 300 amino acids in length.

17. The fusion polynucleotide according to embodiment 16, wherein one of the one or more regions of the surfactant-associated protein D is 80 amino acids to 260 amino acids in length. 18. The fusion polynucleotide according to embodiment 17, wherein one of the one or more regions of the surfactant-associated protein D is 95 amino acids to 160 amino acids in length.

19. The fusion polynucleotide according to embodiment 18, wherein one of the one or more regions of the surfactant-associated protein D is 105 amino acids in length.

20. The fusion polynucleotide according to embodiment 18, wherein one of the one or more regions of the surfactant-associated protein D is 151 amino acids in length.

21. The fusion polynucleotide according to embodiment 17, wherein one of the one or more regions of the surfactant-associated protein D is 256 amino acids in length.

22. The fusion polynucleotide according to any one of embodiments 23-29, wherein the one of the one or more regions of the surfactant-associated protein D is located at the N-terminus of the region of SARS-CoV-2 spike protein.

23. The fusion polynucleotide according to any one of embodiments 15-22, wherein one of the one or more regions of the surfactant-associated protein D is located at the C-terminus of the region of SARS-CoV-2 spike protein.

24. The fusion polynucleotide according to any one of embodiments 15-21, wherein one of the one or more regions of the surfactant-associated protein D is located at the N-terminus of the SARS-CoV-2 RNA-dependent RNA polymerase.

25. The fusion polynucleotide according to any one of embodiments 15-21 and 24, wherein one of the one or more regions of the surfactant-associated protein D is located at the C- terminus of the SARS-CoV-2 RNA-dependent RNA polymerase.

26. The fusion polynucleotide according to embodiment 23 or 25, wherein one of the one or more regions of the surfactant-associated protein D is located at the C-terminus of the 2A self-cleaving peptide.

27. The fusion polynucleotide according to any one of embodiments 1-3, 9, 15-23 and 26, wherein the region of the soluble CD40 ligand is located at the C-terminus of the region of the SARS-CoV-2 spike protein.

28. The fusion polynucleotide according to embodiment 1-3, 9 and 13-23, 26 and 27, wherein the region of the soluble CD40 ligand is located at the C-terminus of one of the one or more regions of the surfactant-associated protein D. 29. The fusion polynucleotide according to any one of embodiments 9 and 12-21, and 24-26, wherein the soluble CD40 ligand is located at the C-terminus of the region of the SARS- CoV-2 RNA-dependent RNA polymerase.

30. The fusion polynucleotide according to any one of embodiments 9 and 12-29, wherein the soluble CD40 ligand is located at the C-terminus of the region of the one of the one or more region of the surfactant-associated protein D.

31. The fusion polynucleotide according to any one of embodiments 1-3, 9 and 12-30, wherein the region of soluble CD40 ligand is 200 to 230 amino acids in length.

32. The fusion polynucleotide according to embodiment 31, wherein the region of the soluble CD40 ligand is 214 amino acids in length.

33. The fusion polynucleotide according to any one of embodiments 4-7, wherein the cellbound CD40 ligand is located at the C-terminus of the region of the SARS-CoV-2 spike protein.

34. The fusion polynucleotide according to any one of embodiments 4-7, wherein the cellbound CD40 ligand is located at the C-terminus of the 2A self-cleaving peptide.

35. The fusion polynucleotide according to embodiment 33 or 34, wherein the cell-bound CD40 ligand is 230 to 260 amino acids in length.

36. The fusion polynucleotide according to embodiment 35, wherein the cell-bound CD40 ligand is 260 amino acids in length.

37. The fusion polynucleotide according to embodiment 35, wherein the cell-bound CD40 ligand is 247 amino acids in length.

38. The fusion polynucleotide according to embodiment 8, wherein the Interferon-a protein is located at the C-terminus of the SARS-CoV-2 spike protein.

39. The fusion polynucleotide according to embodiment 11, wherein the Interferon-a protein is located at the C-terminus of the SARS-CoV-2 RNA-dependent RNA polymerase.

40. The fusion polynucleotide according to any one of embodiments 8, 14, 38 or 39, wherein the Interferon-a protein is located at the C-terminus of the 2A self-cleaving peptide.

41. The fusion polynucleotide according to any one of embodiments 38-40, wherein the Interferon-a protein is 189 to 190 amino acids in length. 42. The fusion polynucleotide according to embodiment 41, wherein the Interferon-a protein is 190 amino acids in length.

43. The fusion polynucleotide according to any one of embodiments 2, 4-8, and 14-42, wherein the 2A self-cleaving peptide is 18 to 23 amino acids in length.

44. The fusion polynucleotide according to embodiment 43, wherein the 2A self-cleaving peptide is 22 amino acids in length.

45. The fusion polynucleotide according to any one of embodiments 10-21, 24-26, 29-32, and 39-44, wherein the Tissue-type Plasminogen Activator protein is an N-terminal region of that protein.

46. The fusion polynucleotide according to embodiment 10-21, 24-26, 29-32, and 39-45, wherein the Tissue-type Plasminogen Activator protein is located at the N-terminus of the SARS-CoV-2 RNA-dependent RNA polymerase.

47. The fusion polynucleotide according to any one of embodiments 1-8, 15-23, 26-28, 31- 38, and 40-44, wherein the region of the SARS-CoV-2 spike protein is 1,100 amino acids to 1,300 amino acids in length.

48. The fusion polynucleotide according to embodiment 47, wherein the region of the SARS- CoV-2 spike protein is 1,192 amino acids in length.

49. The fusion polynucleotide according to embodiment 47, wherein the region of the SARS- CoV-2 spike protein is 1,208 amino acids in length.

50. The fusion polynucleotide according to embodiment 47, wherein the region of the SARS- CoV-2 spike protein is 1,273 amino acids in length.

51. The fusion polynucleotide according to any one of embodiments 1-50, wherein the fusion polynucleotide is encapsulated in the lipid nanoparticle.

52. The fusion polynucleotide according to any one of embodiments 1-8, 15-23, 26-28, 30- 38, 40-44, and 47-51, wherein the fusion polynucleotide encodes a soluble SARS-CoV-2 spike protein.

53. The fusion polynucleotide according to any one of embodiments 1-8, 15-23, 26-28, 30- 38, 40-44, and 47-51, wherein the fusion polynucleotide encodes a cell-bound SARS-CoV-2 spike protein . 54. The fusion polynucleotide according to any one of embodiments 9-21, 24-26, 29-32, 39-

46 and 51, wherein the fusion polynucleotide encodes a soluble SARS-CoV-2 RNA- dependent RNA polymerase.

55. The fusion polynucleotide according to any one of embodiments 9-21, 24-26, 29-32, 39-

46 and 51, wherein the fusion polynucleotide encodes a cell-bound SARS-CoV-2 RNA- dependent RNA polymerase.

56. The fusion polynucleotide according to embodiment 52 or 54, wherein the soluble protein encoded by the fusion polynucleotide is transiently expressed.

57. The fusion polynucleotide according to embodiment 53 or 55, wherein the cell-bound protein encoded by the fusion polynucleotide is transiently expressed.

58. The fusion polynucleotide according to any one of embodiments 1-8, 15-23, 26-28, 30-

38, 40-44, 47-53 and 56-57, wherein the mRNA encoding a region of a SARS-CoV-2 spike protein is a modified mRNA.

59. The fusion polynucleotide according to any one of embodiments 9-21, 24-26, 29-32, 39-

46, 51, and 54-57, wherein the mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase is a modified mRNA.

60. The fusion polynucleotide according to any one of embodiments 1, 2, 9, 13-32, and 40-

59, wherein the mRNA encoding one or more regions of a surfactant-associated protein D is a modified mRNA.

61. The fusion polynucleotide according to any one of embodiments 1-3, 9, 12-33, 38, and 40-60, wherein the mRNA encoding a region of a soluble CD40 ligand is a modified mRNA.

62. The fusion polynucleotide according to any one of embodiments 4-7, 33-38, 43, 44, 47- 53, and 56-58, wherein the mRNA encoding the cell-bound CD40 ligand or is a modified mRNA.

63. The fusion polynucleotide according to any one of embodiments 8, 11-14, and 38-62, wherein the mRNA encoding the Interferon-a protein is a modified mRNA.

64. The fusion polynucleotide according to any one of embodiments 2, 4-8, and 14-63 , wherein the mRNA encoding the 2A self-cleavage peptide is a modified mRNA. 65. The fusion polynucleotide according to any one of embodiments 10-14, 28-32, 39-46, 51, 54-57, and 59-64, wherein the Tissue-type Plasminogen Activator protein is a modified mRNA.

66. The fusion polynucleotide according to any one of embodiments 58-65, wherein the modified mRNA is characterized by one or more uridines modified to pseudouridine.

67. The fusion polynucleotide according to any one of embodiments 1-8, 15-23, 26-28, 30- 38, 40-44, 47-53, 56-58 and 60-66, wherein a variant of the SARS-CoV-2 spike protein is selected from the group consisting of an alpha variant, a beta variant, a gamma variant, a delta variant, an omicron variant, an omicron BA.1 variant, an omicron BA.2 variant, and a deltacron variant.

68. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

47.

69. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

48.

70. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

49.

71. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

50.

72. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

52.

73. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

53.

74. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

55.

75. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

56.

76. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 77. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

58.

78. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

59.

79. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

60.

80. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

61.

81. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

63.

82. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

64.

83. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

65.

84. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

66.

85. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

67.

86. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

68.

87. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

69.

88. A lipid nanoparticle comprising a fusion polypeptide of any one of embodiments 1-87.

89. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 24.

90. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 25. 91. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 26.

92. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 27.

93. A lipid nanoparticle comprising a polynucleotide, wherein the nucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 28.

94. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 29.

95. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 30.

96. A lipid nanoparticle comprising a polynucleotide, wherein the nucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 31.

97. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 32.

98. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 33.

99. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 34.

100. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 35. 101. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 36.

102. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 37.

103. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 38.

104. A lipid nanoparticle comprising a polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 39.

105. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 40.

106. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 41.

107. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 42.

108. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 43.

109. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 44.

110. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 45. 111. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 46.

112. The lipid nanoparticle according to any one of embodiments 88-111, wherein the lipid nanoparticle is capable of inducing a Thl immune response on administration to a subject.

113. The lipid nanoparticle according to embodiment 112, wherein the Thl immune response is a CD8+ T cell stimulated Interferon-gamma (IFN-y) response.

114. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a first region of a surfactant-associated protein D, a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding a second region of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand.

115. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding a region of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand.

116. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a region of a surfactant-associated protein D, a mRNA encoding a region of a SARS-CoV-2 spike protein, and a mRNA encoding a region of a soluble CD40 ligand

117. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a region of a SARS-CoV-2 spike protein and a mRNA encoding a region of a soluble CD40 ligand.

118. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a region of a surfactant-associated protein D and a mRNA encoding a region of a soluble CD40 ligand.

119. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding a 2A self-cleaving peptide, a mRNA encoding a region of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand.

120. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding a 2A self-cleaving peptide, and a mRNA encoding a cell-bound CD40 ligand. 121. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding a 2A self-cleaving peptide, and a mRNA encoding a cell-bound CD40 ligand.

122. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding a 2A self-cleaving peptide, and a mRNA encoding a cell-bound CD40 ligand comprising a deletion in the amino acid sequence of the full-length protein extending from amino acid residue 110 to amino acid residue 122.

123. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding a 2A self-cleaving peptide, and a mRNA encoding an Interferon-a protein.

124. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a region of a surfactant-associated protein D, a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, and a mRNA encoding a region of a soluble CD40 ligand.

125. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a first region of a surfactant-associated protein D, a mRNA encoding a SARS-CoV- 2 spike protein, a mRNA encoding a second region of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand.

126. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a Tissue-type Plasminogen Activator protein, a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, a mRNA encoding a region of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand.

127. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a Tissue-type Plasminogen Activator protein, a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, a mRNA encoding a 2A self-cleavage peptide, a mRNA encoding a region of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand.

128. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a Tissue Plasminogen Activator, a mRNA encoding a SARS-CoV-2 RNA- dependent RNA polymerase, and a mRNA encoding a region of a soluble CD40 ligand. 129. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a Tissue Plasminogen Activator and a mRNA encoding a SARS-CoV-2 RNA- dependent RNA polymerase.

130. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a Tissue Plasminogen Activator, a mRNA encoding a SARS-CoV-2 RNA- dependent RNA polymerase, a mRNA encoding a 2A self-cleavage peptide, and a mRNA encoding an Interferon-a protein.

131. The lipid nanoparticle according to any one of embodiments 88-130, wherein the lipid nanoparticle comprises one or more lipids.

132. The lipid nanoparticle according to embodiment 131, wherein the one or more lipids comprises a condensing lipid, a coating lipid, a structural lipid, or combinations thereof.

133. The lipid nanoparticle according to embodiment 132, wherein the structural lipid is selected from the group consisting of hydrogenated soy phosphatidylcholine, fully hydrogenated soy phosphatidylcholine (HSPC), l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), and cholesterol.

134. The lipid nanoparticle according to embodiment 132, wherein the coating lipid is a PEGylated lipid or a poly(2-oxazoline)-conjugated lipid.

135. The lipid nanoparticle according to embodiment 134, wherein the PEGylated lipid is selected from the group consisting of l,2-dimyristoylrac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)-2000] (DSPE-PEG2000).

136. The lipid nanoparticle according to embodiment 132, wherein the condensing lipid is a cationic lipid or an ionizable lipid.

137. The lipid nanoparticle according to embodiment 136, wherein the cationic lipid is selected from the group consisting of l,2-dioleoyl-3 -trimethylammoniumpropane, 1,2- dioleoyl-3-trimethylammoniumpropane (chloride salt) (DOTAP), dimethyldioctadecylammonium, and dimethyldioctadecylammonium (bromide salt).

138. The lipid nanoparticle according to embodiment 136, wherein the ionizable lipid is heptatriaconta-6, 9, 28, 3 l-tetraen-19-yl 4-(dimethylamino) butanoate (Dlin-MC3-DMA or MC3). 139. A pharmaceutical composition comprising a fusion polynucleotide according to any one of embodiments 1-87 or a lipid nanoparticle according to any one of embodiments 88-138 and a pharmaceutically acceptable carrier.

140. The pharmaceutical composition according to embodiment 139, wherein the pharmaceutically acceptable carrier is an aqueous solution.

141. The pharmaceutical composition according to embodiment 139 or 140, wherein the pharmaceutical composition comprises one or more excipients.

142. The pharmaceutical composition according to embodiment 141, wherein the one or more excipients are selected from the group consisting of a buffer and a pH adjusting agent.

143. The pharmaceutical composition according to embodiment 142, wherein the buffer is selected from the group consisting of citrate, sodium citrate tribasic dehydrate, phosphate- buffered saline, and tris (hydroxymethyl) aminomethane (TRIS-base).

144. The pharmaceutical composition according to embodiment 142, wherein the pH adjusting agent is citric acid.

145. A method for treating or preventing a SARS-CoV-2 infection comprising administering an effective amount of the lipid nanoparticle of any one of embodiments 88-138 or the pharmaceutical composition of any one of embodiments 139-144 to a subject in need or at risk thereof.

146. A method for stimulating an adaptive immune response comprising administering the lipid nanoparticle of any one of embodiments 88-138 or the pharmaceutical composition of any one of embodiments 139-144 to a subject in need thereof.

147. The method according to embodiment 146, wherein the adaptive immune response is characterized by CD8+ T cell activation.

148. The method according to embodiment 146 or 147, wherein the adaptive immune response is characterized by a CD8+ T cell IFN-y response.

149. A method for stimulating a Thl immune response comprising administering the lipid nanoparticle of any one of embodiments 88-138 or the pharmaceutical composition of any one of embodiments 139-144 to a subject in need thereof. 150. The method according to embodiment 149, wherein the Thl immune response is characterized by a lack of IgG production.

151. The method according to embodiment 149 or 150, wherein the Thl immune response is characterized by a downstream CD8+ T cell induction.

152. The method according to any one of embodiments 149-151, wherein the Thl immune response is characterized by a downstream CD8+ T cell IFN-y response.

153. A method for stimulating a CD8+ T cell IFN-y response comprising administering the lipid nanoparticle of any one of embodiments 88-138 or the pharmaceutical composition of any one of embodiments 139-144 to a subject in need thereof.

154. The method according to any one of embodiments 145-153, wherein the lipid nanoparticle of any one of embodiments 88-138 or the pharmaceutical composition of any one of embodiments 139-144 is administered intravenously, intramuscularly, subcutaneously, or intraperitoneally.

155. The method according to any one of embodiments 145-154, wherein the subject is a mammal.

156. The method according to embodiment 155, wherein the subject is a human.

BRIEF DESCRIPTION OF DRAWINGS

[0007] Figures 1A, IB, 1C, and ID show schematics of mRNA encoded polypeptides of various lipid nanoparticle (“LNP”)-based mRNA vaccine embodiments of this disclosure and LNP -based mRNA constructs useful as controls herein. Figure 1A: Panel a shows a polypeptide comprising two regions of a surfactant-associated protein D (SPD), a region of a SARS-CoV-2 Spike protein, and a region of a soluble CD40 ligand (sCD40L); Panel b shows a polypeptide comprising a region of a SARS-CoV-2 Spike protein, a region of an SPD and a region of a sCD40L; and Panel c shows a polypeptide comprising a region of a SARS-CoV-2 Spike protein and a region of a sCD40L. Figure IB: Panel d shows a polypeptide comprising a region of an SPD, a region of a SARS-CoV-2 Spike protein, and a region of a sCD40L; Panel e shows a control polypeptide comprising a region of an SPD and a region of a sCD40L; Panel f shows a control polypeptide comprising a full-length SARS- CoV-2 Spike protein; and Panel g shows a control polypeptide comprising a region of the SARS-CoV-2 Spike protein. Figure 1C: Panel h shows a polypeptide comprising a full- length SARS-Cov-2 Spike protein, a 2 A peptide derived from a porcine teschovirus-1 protein (P2A), a region of an SPD, and a region of a sCD40L; Panel i shows a polypeptide comprising a full-length SARS-CoV-2 Spike protein, a P2A, and a cell-bound CD40 ligand (CD40L), and Panel j shows a polypeptide comprising a full-length SARS-CoV-2 Spike protein, a P2A, and a cell-bound CD40L with a deletion at amino acid residues 110-122 of the full-length CD40L protein (CD40L non-cleavable (“nc”)). Figure ID: Panel k shows a polypeptide comprising a region of a SARS-CoV-2 Spike protein, a P2A, a region of a SPD, and a region of a sCD40L; Panel 1 shows a polypeptide comprising a region of a SARS-CoV- 2 Spike protein, a P2A and a CD40L; and Panel m shows a polypeptide comprising a region of a SARS-CoV-2 Spike protein, a P2A, and a CD40L nc.

[0008] Figure 2 shows schematics of RNA encoded polypeptides of various LNP -based mRNA vaccine embodiments of this disclosure and LNP -based mRNA constructs useful as controls herein. Panel a shows a polypeptide comprising a region of a SARS-CoV-2 Spike protein, a 2 A peptide derived from a porcine teschovirus-1 protein (P2A), and an Interferon-a protein (IFNa). Panel b shows a polypeptide comprising a full-length SARS-CoV-2 Spike protein, a P2A, and an IFNa. Panel c shows a control polypeptide comprising an IFNa.

[0009] Figures 3A and 3B show schematics of RNA encoded polypeptides of various lipid nanoparticle-based mRNA vaccine embodiments of this disclosure and lipid nanoparticlebased mRNA constructs useful as controls herein. Figure 3A: Panel a shows a polypeptide comprising a region of a surfactant-associated protein D (SPD), a SARS-CoV-2 RNA- dependent RNA polymerase protein (SARS-CoV-2 nspl2), and a region of a soluble CD40 ligand (sCD40L); Panel b shows a polypeptide comprising two regions of an SPD, a SARS- CoV-2 nspl2, and a region of a sCD40L; Panel c shows a polypeptide comprising a region of a Tissue-type Plasminogen Activator protein (TP A), a SARS-CoV-2 nspl2, a region of an SPD, and a region of an sCD40L; and Panel d shows a polypeptide comprising a region of a TP A, a SARS-CoV-2 nspl2, a 2A peptide derived from a porcine teschovirus-1 protein (P2A), a region of an SPD, and a region of a sCD40L. Figure 3B: Panel e shows a polypeptide comprising a region of a TP A, a SARS-CoV-2 nspl2, and a region of a sCD40; Panel f shows a polypeptide comprising a region of a TPA and a SARS-CoV-2 nspl2; and Panel g shows a polypeptide comprising a region of a TPA, a SARS-COV-2 nspl2, a P2A, and an IFNa. [0010] Figure 4 shows a graph of body weight change (%) over time (days post-treatment start) in mice that were injected with different types of mRNA encapsulated in lipid nanoparticles or a diluent control.

[0011] Figure 5 shows a graph of SARS-CoV-2 total spike IgG-specific interpolated titer points measured at an optical density of 0.3 at 450 nm (OD450) over time (days posttreatment start) in mice that were injected with different types of mRNA encapsulated in lipid nanoparticles and controls of this disclosure or a diluent control.

[0012] Figures 6A and 6B show graphs quantifying the numbers of splenocytes extracted from mice treated with mRNA encapsulated lipid nanoparticles (LNPs) and controls of this disclosure that secrete Interferon-y (IFN-y) after stimulation with a SARS-CoV-2 Spikespecific peptide mix (Figure 6A) or with concanavalin A (positive control) (Figure 6B) using an ELISpot assay.

[0013] Figures 7A and 7B show graphs quantifying the numbers of splenocytes extracted from mice treated with mRNA encapsulated lipid nanoparticles (LNPs) and controls of this disclosure that secrete Interleukin-4 (IL-4) after stimulation with a SARS-CoV-2 Spikespecific peptide mix (Figure 7A) or with concanavalin A (positive control) (Figure 7B) using an ELISpot assay.

[0014] Figure 8 shows a graph of an IC50 of a Plaque Reduction Neutralization Test (PRNT) using day 49 serum from mice treated with the various mRNA encapsulated lipid nanoparticles (LNPs) and controls of this disclosure.

[0015] Figure 9 shows SARS-CoV-2 Spike expression status (soluble or cell-bound) and endosome trapping capability corresponding to the schematics of the encoded polypeptides of various lipid nanoparticle-based mRNA vaccine embodiments of this disclosure and lipid nanoparticle-based mRNA constructs useful as controls herein.

[0016] Figure 10 shows SARS-CoV-2 Spike expression status (soluble or cell-bound) and endosome trapping capability corresponding to the schematics of the encoded polypeptides of various lipid nanoparticle-based mRNA vaccine embodiments of this disclosure and lipid nanoparticle-based mRNA constructs useful as controls herein.

[0017] Figure 11 shows schematics of the cleavage and multimeric formation of encoded polypeptides of various lipid nanoparticle-based mRNA vaccine embodiments of this disclosure and lipid nanoparticle-based mRNA constructs useful as controls herein. [0018] Figures 12 A and 12B shows schematics of encoded polypeptides of various lipid nanoparticle-based mRNA vaccine embodiments of this disclosure and lipid nanoparticlebased mRNA constructs useful as controls herein (Figure 12A) and cleavage of the encoded polypeptides (Figure 12B).

[0019] Figures 13 A and 13B shows schematics of encoded polypeptides of various lipid nanoparticle-based mRNA vaccine embodiments of this disclosure and lipid nanoparticlebased mRNA constructs useful as controls herein (Figure 13A) and cleavage of the encoded polypeptides (Figure 13B).

DETAILED DESCRIPTION

[0020] The present disclosure provides lipid nanoparticles comprising fusion RNA polynucleotides encoding polypeptides, pharmaceutical compositions comprising the lipid nanoparticles, methods for treating SARS-CoV-2 infection and methods for activating Thl T cells and CD8 cytotoxic T cells and, thus, a cellular immune response in a subject suffering from or at risk for SARS-CoV-2 infection.

Definitions

[0021] The term “herein” means the entire application.

[0022] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. In case of conflict, the present specification, including definitions, will control.

[0023] It should be understood that any of the embodiments described herein, including those described under different aspects of the disclosure and different parts of the specification can be combined with one or more other embodiments of this disclosure, unless explicitly disclaimed or improper. Combination of embodiments are not limited to those specific combinations recited in the various multiple dependent embodiments herein.

[0024] All of the publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

[0025] Throughout this specification and embodiments, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

[0026] The term “including,” as used herein, means “including but not limited to.” “Including” and “including but not limited to” are used interchangeably. Thus, these terms will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

[0027] Any example(s) following the term “e.g.” or “for example” is not meant to be exhaustive or limiting.

[0028] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0029] The articles "a", "an" and “the” 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.

[0030] In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

[0031] The term “SARS-CoV-2 spike protein,” “spike protein,” “Spike,” and “S protein” as used interchangeably herein refers to a SARS-CoV-2 spike protein of the Wuhan strain of the virus or variants thereof or the spike protein or variants thereof of other variants of the virus. In some embodiments, the SARS-CoV-2 spike protein is the full-length 1273 amino acid protein of the Wuhan strain of the virus or the corresponding residues in other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1192 amino acids of the Wuhan strain of the virus, i.e., with the signal sequence and the transmembrane domain being deleted, or the corresponding residues in other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is a polypeptide extending from amino acid residue 17 to residue 1208 (1192 amino acids) of the spike protein of the Wuhan strain of the virus or the corresponding residues in other variants of the virus. In some embodiments, a region of the SARS-CoV-2 spike protein of this disclosure is 1208 amino acids, z.e., with the transmembrane domain being deleted, or the corresponding residues in other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is a polypeptide extending from amino acid residue 1 to residue 1208 of the spike protein of the Wuhan strain of the virus or the corresponding residues in other variants of the virus. In some embodiments, a SARS-CoV-2 spike protein region of this disclosure refers to a SARS-CoV- 2 spike protein with the transmembrane domain deleted. In some embodiments, a SARS- CoV-2 spike protein region of this disclosure refers to a SARS-CoV-2 spike protein with one or both of the transmembrane domain and the signal peptide deleted. In some embodiments, a SARS-CoV-2 spike protein region of this disclosure refers to a SARS-CoV-2 spike protein having one or more amino acid deletions in the Spike protein. In some embodiments, a SARS-CoV-2 spike protein region refers to a SARS-CoV-2 spike protein having one or more amino acid deletions in the Spike protein and its signal peptide. In some embodiments, a region of a SARS-CoV-2 spike protein is or comprises the amino acid sequence according to SEQ ID NO: 79. In some embodiments, a region of a SARS-CoV-2 spike protein is or comprises the amino acid sequence according to SEQ ID NO: 31. In some embodiments, a SARS-CoV-2 spike protein is or comprises the amino acid sequence according to SEQ ID NO: 28.

[0032] The term “surfactant-associated protein D,” “surfactant protein D” or “SPD” are used interchangeably herein. In some embodiments, an SPD of this disclosure is a mouse SPD. In some embodiments, an SPD of this disclosure is a human SPD. In some embodiments, a region of a surfactant-associated protein D of this disclosure is 105 amino acids in length. In some embodiments, a region of a surfactant-associated protein D of this disclosure extends from amino acid residue 1 to amino acid residue 105 of a mouse SPD or the corresponding amino acid residues in a human SPD. In some embodiments, a region of a surfactant-associated protein D of this disclosure is 151 amino acids in length. In some embodiments, a region of a surfactant-associated protein D of this disclosure extends from amino acid residue 106 to amino acid residue 256 of a mouse SPD or the corresponding amino acid residues in a human SPD. In some embodiments, a region of a surfactant- associated protein D of this disclosure is 256 amino acids in length. In some embodiments, a region of a surfactant-associated protein D of this disclosure extends from amino acid residue 1 to amino acid residue 256 of a mouse SPD or the corresponding amino acid residues in a human SPD. In some embodiments, one or more regions of a surfactant- associated protein D of this disclosure refers to two regions, wherein the first region is 105 amino acids in length and wherein the second region is 151 amino acids in length. In some embodiments, one or more regions of a surfactant-associated protein D of this disclosure refers to two regions, wherein the first region extends from amino acid residue 1 to amino acid residue 105 of a mouse SPD or the corresponding amino acid residues in a human SPD and wherein the second region extends from amino acid residue 106 to amino acid residue 256 of a mouse SPD or the corresponding amino acid residues in a human SPD. In some embodiments, one or more regions of a surfactant-associated protein D of this disclosure refers to two regions, wherein the second region enables trapping of the encoded polypeptide in the endosome. In some embodiments, one or more regions of a surfactant-associated protein D of this disclosure refers to one region that has multimerization activity but not does not enable trapping of the encoded polypeptide in the endosome. In some embodiments, the one or more regions of a surfactant-associated protein D of this disclosure refers to one region that enables aggregation of a soluble CD40 ligand. In some embodiments, a region of a surfactant-associated protein D of this disclosure is or comprises the amino acid sequence according to SEQ ID NO: 71. In some embodiments, the one or more regions of a surfactant-associated protein D of this disclosure refers to one region that enables aggregation of a soluble CD40 ligand. In some embodiments, a region of a surfactant- associated protein D of this disclosure is or comprises the amino acid sequence according to SEQ ID NO: 73. In some embodiments, the one or more regions of a surfactant-associated protein D of this disclosure refers to one region that enables aggregation of a soluble CD40 ligand. In some embodiments, a region of a surfactant-associated protein D of this disclosure is or comprises the amino acid sequence according to SEQ ID NO: 75.

[0033] The terms “CD40 ligand” and “CD40L” as used interchangeably herein refers to a cell-bound CD40 ligand. In some embodiments, CD40L is a full-length cell-bound CD40L. In some embodiments, a CD40L is a mouse CD40L. In some embodiments, a CD40L is a human CD40L. In some embodiments, a CD40L is characterized by a deletion from amino acid residue 110 to amino acid residue 122 (“Al 10-122”) of a mouse CD40L or the corresponding amino acid residues in a human CD40L and is non-cleavable (also referred to as “CD40L nc”). In some embodiments, a CD40L is or comprises an amino acid sequence according to SEQ ID NO: 84. In some embodiments, a CD40L nc is or comprises an amino acid sequence according to SEQ ID NO: 86.

[0034] The terms “soluble CD40 ligand” and “sCD40L” are used interchangeably herein. In some embodiments, a sCD40L is a mouse sCD40L. In some embodiments, a sCD40L is a human sCD40L. In some embodiments, a region of a sCD40L of this disclosure is the extracellular domain. In some embodiments, a region of a sCD40L of this disclosure extends from amino acid residue 47 to amino acid residue 260 of a mouse sCD40L or the corresponding amino acid residues in a human sCD40L. In some embodiments, a sCD40L- SPD polypeptide of this disclosure forms a multimeric protein. In some embodiments, a sCD40L-SPD polypeptide of this disclosure forms a tetrameric protein. In some embodiments, a region of a sCD40L is or comprises the amino acid sequence according to SEQ ID NO: 77.

[0035] The terms “Tissue-type Plasminogen Activator” and “tPA” and regions thereof as used interchangeably herein refers to a tPA signal peptide domain. In some embodiments, a tPA is a human tPA. In some embodiments, a tPA signal peptide domain of this disclosure is 22 amino acids in length. In some embodiments, a tPA signal peptide of this disclosure extends from amino acid residue 1 to amino acid residue 22. In some embodiments, a tPA is or comprises the amino acid sequence according to SEQ ID NO: 90.

[0036] The terms “SARS-CoV-2 RNA-dependent RNA polymerase,” “SARS-CoV-2 nspl2,” and “nspl2” are used interchangeably herein. In some embodiments, SARS-CoV-2 nspl2 functions as an antigen. In some embodiments, a SARS-CoV-2 RNA-dependent RNA polymerase is or comprises an amino acid sequence according to SEQ ID NO: 88.

[0037] The terms “2 A peptide” and “2 A self-cleaving peptide” are used interchangeably here. In some embodiments, a 2A self-cleaving peptide of this disclosure is or comprises an amino acid sequence according to SEQ ID NO: 82.

[0038] The terms “Interferon-a” and “IFNa” are used interchangeably herein. In some embodiments, an IFNa is or comprises an amino acid sequence according to SEQ ID NO: 39.

[0039] The terms “Interferon-y” and “IFNy” are used interchangeably herein. [0040] As used herein, the term “region” refers to a domain of a protein, a combination of two or more domains of a protein, or a protein with partial or complete deletions of one or more domains.

[0041] Where aspects or embodiments are described in terms of a Markush group or other grouping of alternatives, the present application encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members.

[0042] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the various aspects and embodiments of this disclosure. The materials, methods, and examples are illustrative only and not intended to be limiting.

Polynucleotide Constructs and Encoded Polypeptides

[0043] In some embodiments, the fusion RNA polynucleotide of this disclosure comprises a mRNA encoding a SARS-CoV-2 spike protein or a region thereof, a mRNA encoding one or more regions of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding one or more regions of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a SARS-CoV-2 spike protein, a mRNA encoding one or more regions of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a SARS-CoV-2 spike protein or a region thereof, a mRNA encoding one or more regions of a surfactant-associated protein D, a mRNA encoding a 2A self-cleaving peptide and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding one or more regions of a surfactant- associated protein D, a mRNA encoding a 2A self-cleaving peptide and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a SARS-CoV-2 spike protein, a mRNA encoding one or more regions of a surfactant-associated protein D, a mRNA encoding a 2A selfcleaving peptide and a mRNA encoding a region of a soluble CD40 ligand.

[0044] In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a region of a SARS-CoV-2 spike protein and a mRNA encoding a region of a soluble CD40 ligand.

[0045] In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a SARS-CoV-2 spike protein or a region thereof, a mRNA encoding a cellbound CD40 ligand, and a mRNA encoding a 2A self-cleaving peptide. In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding a cell-bound CD40 ligand, and a mRNA encoding a 2A self-cleaving peptide. In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a SARS-CoV-2 spike protein, a mRNA encoding a cell-bound CD40 ligand, and a mRNA encoding a 2A self-cleaving peptide. In some embodiments, the cell-bound CD40 ligand of this disclosure is characterized by the amino acid sequence of the full-length cell -bound CD40 ligand. In some embodiments, the mRNA encoding a cell-bound CD40 ligand has a deletion of one or more nucleotides. In some embodiments, the cell-bound CD40 ligand of this disclosure has a deletion of 23 amino acids in the amino acid sequence of the full-length cell-bound CD40 ligand. In some embodiments, a cell-bound CD40 ligand of this disclosure has a deletion in the amino acid sequence of the full-length protein from amino acid residue 110 to amino acid residue 122. In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a SARS-CoV-2 spike protein or a region thereof, a mRNA encoding a cell-bound CD40 ligand, and a mRNA encoding a 2A self-cleaving peptide, wherein the mRNA encoding the cell-bound CD40 ligand has a deletion of one or more nucleotides as compared to the mRNA encoding the full-length cell bound protein. In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding a cell-bound CD40 ligand, and a mRNA encoding a 2A self-cleaving peptide, wherein the mRNA encoding the cell-bound CD40 ligand has a deletion of one or more nucleotides as compared to the mRNA encoding the full-length cell bound protein. In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a SARS-CoV-2 spike protein, a mRNA encoding a cell-bound CD40 ligand, and a mRNA encoding a 2A self-cleaving peptide, wherein the mRNA encoding the cell-bound CD40 ligand has a deletion of one or more nucleotides as compared to the mRNA encoding the full-length cell bound protein.

[0046] In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a SARS-CoV-2 spike protein or a region thereof, a mRNA encoding an Interferon-a protein, and a mRNA encoding a 2A self-cleaving peptide. In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding an Interferon-a protein, and a mRNA encoding a 2A self-cleaving peptide. In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a SARS-CoV-2 spike protein, a mRNA encoding an Interferon-a protein, and a mRNA encoding a 2A self-cleaving peptide.

[0047] In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, a mRNA encoding one or more regions of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand.

[0048] In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase and a mRNA encoding a region of a Tissue-type Plasminogen Activator protein. In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a SARS-CoV-2 RNA- dependent RNA polymerase, a mRNA encoding a region of a Tissue-type Plasminogen Activator protein, and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, a mRNA encoding a region of a Tissuetype Plasminogen Activator protein, a mRNA encoding a region of a soluble CD40 ligand, and a mRNA encoding one or more regions of a surfactant-associated protein D. In some embodiments, the fusion polynucleotide of this disclosure comprises a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, a mRNA encoding a region of a Tissuetype Plasminogen Activator protein, a mRNA encoding a region of a soluble CD40 ligand, a mRNA encoding one or more regions of a surfactant-associated protein D, and a mRNA encoding a 2A self-cleaving peptide. [0049] In some embodiments, the fusion polynucleotides of this disclosure further comprise a 2A self-cleaving peptide. In some embodiments, the 2A self-cleaving peptide is the 2 A peptide of the porcine teschovirus-1. In some embodiments, the 2 A self-cleaving peptide is the 2A peptide of the Thosea asigna virus. In some embodiments, the 2A selfcleaving peptide is the 2A peptide of the foot-and-mouth disease virus. In some embodiments, the 2A self-cleaving peptide is the 2A peptide of the equine rhinitis A virus.

[0050] In some embodiments, one or more regions of the surfactant-associated protein D of this disclosure is an N-terminal region of that protein. In some embodiments, one of the one or more regions of the surfactant-associated protein D is located at the N-terminus of the region of SARS-CoV-2 spike protein. In some embodiments, one of the one or more regions of the surfactant-associated protein D of this disclosure is located at the C-terminus of the region of SARS-CoV-2 spike protein. In some embodiments, one of the one or more regions of the surfactant-associated protein D of this disclosure is located at the N-terminus of the SARS-CoV-2 RNA-dependent RNA polymerase. In some embodiments, one of the one or more regions of the surfactant-associated protein D of this disclosure is located at the C- terminus of the SARS-CoV-2 RNA-dependent RNA polymerase. In some embodiments, one of the one or more regions of the surfactant-associated protein D of this disclosure is located at the C-terminus of the 2A self-cleaving peptide.

[0051] In some embodiments, one of the one or more regions of the surfactant-associated protein D is 50 amino acids to 300 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 80 amino acids to 260 amino acids in length. In some embodiments, one of the one or more regions of the surfactant- associated protein D is 95 amino acids to 160 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 95 amino acids to 115 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 130 amino acids to 170 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 140 amino acids to 160 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 225 amino acids to 275 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 245 amino acids to 265 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 100 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 101 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 102 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 103 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 104 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 105 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 106 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 107 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 108 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 109 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 110 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 145 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 146 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 147 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 148 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 149 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 150 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 151 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 152 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 153. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 154. In some embodiments, one of the one or more regions of the surfactant- associated protein D is 155. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 156. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 157. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 158. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 159. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 160. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 250 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 251 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 252 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 253 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 254 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 255 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 256 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 257 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 258 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 259 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 260 amino acids in length. In some embodiments, one of the one or more regions of the surfactant-associated protein D is 261 amino acids in length.

[0052] In some embodiments, the region of the soluble CD40 ligand of this disclosure is located at the C-terminus of the region of the SARS-CoV-2 spike protein. In some embodiments, the region of the soluble CD40 ligand of this disclosure is located at the C- terminus of one of the one or more regions of the surfactant-associated protein D. In some embodiments, the soluble CD40 ligand of this disclosure is located at the C-terminus of the region of the SARS-CoV-2 RNA-dependent RNA polymerase. In some embodiments, the soluble CD40 ligand of this disclosure is located at the C-terminus of the region of the one of the one or more region of the surfactant-associated protein D.

[0053] In some embodiments, the region of soluble CD40 ligand is 200 to 230 amino acids in length. In some embodiments, the region of soluble CD40 ligand is 210 to 220 amino acids in length. In some embodiments, the region of the soluble CD40 ligand is 210 amino acids in length. In some embodiments, the region of the soluble CD40 ligand is 211 amino acids in length. In some embodiments, the region of the soluble CD40 ligand is 212 amino acids in length. In some embodiments, the region of the soluble CD40 ligand is 213 amino acids in length. In some embodiments, the region of the soluble CD40 ligand is 214 amino acids in length. In some embodiments, the region of the soluble CD40 ligand is 215 amino acids in length. In some embodiments, the region of the soluble CD40 ligand is 216 amino acids in length. In some embodiments, the region of the soluble CD40 ligand is 217 amino acids in length. In some embodiments, the region of the soluble CD40 ligand is 218 amino acids in length. In some embodiments, the region of the soluble CD40 ligand is 219 amino acids in length. In some embodiments, the region of the soluble CD40 ligand is 220 amino acids in length.

[0054] In some embodiments, the cell-bound CD40 ligand of this disclosure is located at the C-terminus of the region of the SARS-CoV-2 spike protein. In some embodiments, the cell-bound CD40 ligand of this disclosure is located at the C-terminus of the 2A self-cleaving peptide.

[0055] In some embodiments, the cell-bound CD40 ligand is 230 to 260 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 240 to 255 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 240 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 241 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 242 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 243 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 244 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 245 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 246 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 247 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 248 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 249 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 250 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 251 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 252 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 253 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 254 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 255 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 256 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 257 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 258 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 259 amino acids in length. In some embodiments, the cell-bound CD40 ligand is 260 amino acids in length. [0056] In some embodiments, the Interferon-a protein of this disclosure is located at the C- terminus of the SARS-CoV-2 spike protein. In some embodiments, the Interferon-a protein of this disclosure is located at the C-terminus of the SARS-CoV-2 RNA-dependent RNA polymerase. In some embodiments, the Interferon-a protein of this disclosure is located at the C-terminus of the 2A self-cleaving peptide.

[0057] In some embodiments, the Interferon-a protein is 189 to 190 amino acids in length (including signal sequence). In some embodiments, the Interferon-a protein is 189 amino acids in length. In some embodiments, the Interferon-a protein is 190 amino acids in length.

[0058] In some embodiments, the 2A self-cleaving peptide is 18 to 23 amino acids in length. In some embodiments, the 2A self-cleaving peptide is 18 amino acids in length. In some embodiments, the 2A self-cleaving peptide is 19 amino acids in length. In some embodiments, the 2A self-cleaving peptide is 20 amino acids in length. In some embodiments, the 2 A self-cleaving peptide is 21 amino acids in length. In some embodiments, the 2A self-cleaving peptide is 22 amino acids in length. In some embodiments, the 2A self-cleaving peptide is 23 amino acids in length.

[0059] In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is a polypeptide extending anywhere from amino acid residue 1,100 to amino acid residue 1,300 of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,185 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,186 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,187 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,188 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,189 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,190 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,191 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,192 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,193 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,194 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,195 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,196 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,197 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,198 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,199 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,200 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is a polypeptide extending from amino acid residue 17 to amino acid residue 1208 of the spike protein of the Wuhan strain of the virus or the corresponding residues in other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,201 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues in other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,202 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues in other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,203 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues in other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,204 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues in other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,205 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues in other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,206 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues in other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,207 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues in other variants of the virus. In some embodiments, a region of a SARS-CoV-2 spike protein of this disclosure is 1,208 amino acids of the spike protein of the Wuhan strain of the virus or the corresponding residues in other variants of the virus. In some embodiments, the region of the SARS-CoV-2 spike protein of this disclosure is a polypeptide extending from amino acid residue 1 to amino acid residue 1208 of the spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV-2 spike protein is 1,265 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV-2 spike protein is 1,266 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV-2 spike protein is 1,267 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV- 2 spike protein is 1,268 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV-2 spike protein is 1,269 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV-2 spike protein is 1,270 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV-2 spike protein is 1,271 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV-2 spike protein is 1,272 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV- 2 spike protein is 1,273 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV-2 spike protein is 1,274 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV-2 spike protein is 1,275 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV-2 spike protein is 1,276 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV-2 spike protein is 1,277 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV- 2 spike protein is 1,278 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV-2 spike protein is 1,279 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus. In some embodiments, the SARS-CoV-2 spike protein is 1,280 amino acids of the full-length spike protein of the Wuhan strain of the virus or the corresponding residues of other variants of the virus.

[0060] In some embodiments, the Tissue-type Plasminogen Activator protein is a signal peptide domain. In some embodiments, the Tissue-type Plasminogen Activator protein is located at the N-terminus of the SARS-CoV-2 RNA-dependent RNA polymerase.

[0061] In some embodiments, the fusion polynucleotide of this disclosure encodes a soluble SARS-CoV-2 spike protein. In some embodiments, the fusion polynucleotide of this disclosure encodes a cell-bound SARS-CoV-2 spike protein. In some embodiments, the fusion polynucleotide of this disclosure encodes a soluble SARS-CoV-2 RNA-dependent RNA polymerase. In some embodiments, the fusion polynucleotide of this disclosure encodes a cell-bound SARS-CoV-2 RNA-dependent RNA polymerase.

[0062] In some embodiments, the soluble SARS-CoV-2 spike protein or soluble SARS- CoV-2 RNA-dependent RNA polymerase encoded by the fusion polynucleotide of this disclosure is transiently expressed. In some embodiments, the cell-bound SARS-CoV-2 spike protein or cell-bound SARS-CoV-2 RNA-dependent RNA polymerase encoded by the fusion polynucleotide of this disclosure is transiently expressed. In some embodiments, the soluble SARS-CoV-2 spike protein encoded by the fusion polynucleotide of this disclosure is transiently expressed. In some embodiments, the cell-bound SARS-CoV-2 spike protein encoded by the fusion polynucleotide of this disclosure is transiently expressed. In some embodiments, the soluble SARS-CoV-2 RNA-dependent RNA polymerase encoded by the fusion polynucleotide of this disclosure is transiently expressed. In some embodiments, the cell-bound SARS-CoV-2 RNA-dependent RNA polymerase encoded by the fusion polynucleotide of this disclosure is transiently expressed. In some embodiments, the soluble CD40 ligand or the cell-bound CD40 ligand encoded by the fusion polynucleotide of this disclosure is transiently expressed. In some embodiments, the soluble CD40 ligand encoded by the fusion polynucleotide of this disclosure is transiently expressed. In some embodiments, the cell-bound CD40 ligand encoded by the fusion polynucleotide of this disclosure is transiently expressed.

[0063] In some embodiments, the mRNA encoding a region of a SARS-CoV-2 spike protein is a modified mRNA. In some embodiments, the mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase is a modified mRNA. In some embodiments, the mRNA encoding one or more regions of a surfactant-associated protein D is a modified mRNA. In some embodiments, the mRNA encoding a region of a soluble CD40 ligand is a modified mRNA. In some embodiments, the mRNA encoding the cell-bound CD40 ligand is a modified mRNA. In some embodiments, the mRNA encoding the Interferon-a protein is a modified mRNA. In some embodiments, the mRNA encoding the 2A self-cleavage peptide is a modified mRNA. In some embodiments, the mRNA encoding the Tissue-type Plasminogen Activator protein is a modified mRNA. In some embodiments, the mRNA modification is a chemical mRNA modification. In some embodiments, a mRNA modification is characterized by one or more chemical modification selected from pseudouridine, N1 -methylpseudouridine, N1 -ethylpseudouridine, 2-thiouridine, 4'- thiouridine, 5-methylcytosine, 5 -methyluridine, 2-thio-l -methyl- 1-deaza-pseudouri dine, 2- thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio- dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy- pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyl uridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a pseudouridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a Nl-methyl-pseudouridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 2-thiouridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 2’-O-methyl uridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a N1 -ethylpseudouridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 4'-thiouridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 5-methylcytosine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 5- methyluridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 2-thio-l -methyl- 1-deaza-pseudouri dine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 2-thio-l -methylpseudouridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 2-thio-5-aza-uridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 2-thio-dihydropseudouridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 2- thio-dihydrouridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 2-thio-pseudouridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 4-methoxy-2 -thiopseudouridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 4-methoxy-pseudouridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 4-thio-l-methyl-pseudouridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 4-thio-pseudouridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 5-aza-uridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a dihydropseudouridine. In some embodiments, the modified mRNA is characterized by one or more uridines modified to a 5-methoxyuridine.

[0064] In some embodiments, the SARS-CoV-2 variant is selected from the group consisting of an alpha variant, a beta variant, a gamma variant, a delta variant, an omicron variant, an omicron BA.l variant, an omicron BA.2 variant, a deltacron variant and other variants that may arise in the future. In some embodiments, the SARS-CoV-2 variant is an alpha variant. In some embodiments, the SARS-CoV-2 variant is a beta variant. In some embodiments, the SARS-CoV-2 variant is a gamma variant. In some embodiments, the SARS-CoV-2 variant is a delta variant. In some embodiments, the SARS-CoV-2 variant is an omicron variant. In some embodiments, the SARS-CoV-2 variant is an omicron BA.l sub-variant. In some embodiments, the SARS-CoV-2 variant is an omicron BA.2 subvariant. In some embodiments, the SARS-CoV-2 variant is a deltacron variant.

[0065] In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, the polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, the polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the fusion polynucleotide of this disclosure encodes a protein comprising or being the amino acid sequence set forth in SEQ ID NO: 46.

[0066] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a first region of a surfactant-associated protein D, a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding a second region of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a surfactant-associated protein D characterized by amino acid residues 1-105 of a mouse SPD or the corresponding residues in a human SPD, a mRNA encoding a region of a SARS-CoV-2 spike protein characterized by amino acid residues 17-1208 of the Wuhan strain of the virus or the corresponding residues in other variants of the virus, a mRNA encoding a second region of a surfactant-associated protein D characterized by amino acid residues 106-256 of a mouse SPD or the corresponding residues in a human SPD, and a mRNA encoding a region of a soluble CD40 ligand characterized by amino acid residues 47- 260 of a mouse sCD40L or the corresponding residues in a human sCD40.

[0067] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding a region of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a SARS-CoV-2 spike protein characterized by amino acid residues 1-1208 of the Wuhan strain of the virus or the corresponding residues in other variants of the virus,, a mRNA encoding a region of a surfactant-associated protein D characterized by amino acid residues 106-256 of a mouse SPD or the corresponding residues in a human SPD, and a mRNA encoding a region of a soluble CD40 ligand characterized by amino acid residues 47-260 of a mouse sCD40L or the corresponding residues in a human sCD40L.

[0068] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a surfactant-associated protein D, a mRNA encoding a region of a SARS-CoV-2 spike protein, and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a surfactant-associated protein D characterized by amino acid residues 1-105 of a mouse SPD or the corresponding residues in a human SPD, a mRNA encoding a region of a SARS-CoV-2 spike protein characterized by amino acid residues 17- 1208 of the Wuhan strain of the virus or the corresponding residues in other variants of the virus, and a mRNA encoding a region of a soluble CD40 ligand characterized by amino acid residues 47-260 of a mouse sCD40L or the corresponding residues in a human sCD40.

[0069] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a SARS-CoV-2 spike protein and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a SARS-CoV-2 spike protein characterized by amino acid residues 1-1208 of the Wuhan strain of the virus or the corresponding residues in other variants of the virus and a mRNA encoding a region of a soluble CD40 ligand characterized by amino acid residues 47-260.

[0070] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a surfactant-associated protein D and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the control fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a surfactant-associated protein D characterized by amino acid residues 1-256 of a mouse SPD or the corresponding residues in a human SPD and a mRNA encoding a region of a soluble CD40 ligand characterized by amino acid residues 47-260 of a mouse sCD40L or the corresponding residues in a human sCD40L.

[0071] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a SARS-CoV-2 spike protein, a mRNA encoding a 2A self-cleaving peptide, a mRNA encoding a region of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a SARS- CoV-2 spike protein, a mRNA encoding a 2A self-cleaving peptide, a mRNA encoding a region of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in sequence a mRNA encoding a SARS-CoV-2 spike protein, a mRNA encoding a P2A selfcleaving peptide, a mRNA encoding a region of a surfactant-associated protein D characterized by amino acid residues 1-256 of a mouse SPD or the corresponding residues in a human SPD, and a mRNA encoding a region of a soluble CD40 ligand characterized by amino acid residues 47-260 of a mouse sCD40L or the corresponding residues in a human sCD40L. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a SARS-CoV-2 spike protein characterized by amino acid residues 1-1208 of the Wuhan strain of the virus or the corresponding residues in other variants of the virus, a mRNA encoding a P2A self-cleaving peptide, a mRNA encoding a region of a surfactant-associated protein D characterized by amino acid residues 1-256 of a mouse SPD or the corresponding residues in a human SPD, and a mRNA encoding a region of a soluble CD40 ligand characterized by amino acid residues 47-260 of a mouse sCD40L or the corresponding residues in a human sCD40L.

[0072] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a SARS-CoV-2 spike protein, a mRNA encoding a 2A self-cleaving peptide, and a mRNA encoding a cell-bound CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a SARS- CoV-2 spike protein, a mRNA encoding a 2A self-cleaving peptide, and a mRNA encoding a cell-bound CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a SARS-CoV-2 spike protein characterized by amino acid residues 1-1273 of the Wuhan strain of the virus or the corresponding residues in other variants of the virus, a mRNA encoding a P2A self-cleaving peptide, and a mRNA encoding a cell-bound CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in sequence a mRNA encoding a region of a SARS-CoV-2 spike protein characterized by amino acid residues 1-1208 of the Wuhan strain of the virus or the corresponding residues in other variants of the virus, a mRNA encoding a P2A self-cleaving peptide, and a mRNA encoding a cell-bound CD40 ligand.

[0073] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a SARS-CoV-2 spike protein, a mRNA encoding a 2A self-cleaving peptide, and a mRNA encoding a cell-bound non-cleavable CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding a 2A self-cleaving peptide, and a mRNA encoding a cell-bound non-cleavable CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a SARS-CoV-2 spike protein characterized by amino acid residues 1-1273 of the Wuhan strain of the virus or the corresponding residues in other variants of the virus, a mRNA encoding a P2A self-cleaving peptide, and a mRNA encoding a cell-bound non- cleavable CD40 ligand comprising a deletion in the amino acid sequence of the full-length protein extending from residue 110 to residue 122. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a SARS- CoV-2 spike protein of amino acid position 1-1208 of the Wuhan strain of the virus or the corresponding residues in other variants of the virus, a mRNA encoding a P2A self-cleaving peptide, and a mRNA encoding a cell-bound non-cleavable CD40 ligand comprising a deletion in the amino acid sequence of the full-length protein extending from residue 110 to residue 122.

[0074] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding a 2 A self-cleaving peptide, and a mRNA encoding an Interferon-a protein. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a SARS- CoV-2 spike protein, a mRNA encoding a 2A self-cleaving peptide, and a mRNA encoding an Interferon-a protein. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a SARS-CoV-2 spike protein characterized by amino acid residue 1-1208 of the Wuhan strain of the virus or the corresponding residues in other variants of the virus, a mRNA encoding a P2A self-cleaving peptide, and a mRNA encoding an Interferon-a protein. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a SARS-CoV-2 spike protein characterized by amino acid residues 1-1273 of the Wuhan strain of the virus or the corresponding residues in other variants of the virus, a mRNA encoding a P2A self-cleaving peptide, and a mRNA encoding an Interferon-a protein.

[0075] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a region of a surfactant-associated protein D, a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in sequence a mRNA encoding a region of a surfactant-associated protein D characterized by amino acid residues 1-105 of a mouse SPD or the corresponding residues in a human SPD, a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, and a mRNA encoding a region of a soluble CD40 ligand characterized by amino acid residues 47- 260 of a mouse sCD40L or the corresponding residues in a human sCD40L.

[0076] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a first region of a surfactant-associated protein D, a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, a mRNA encoding a second region of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a first region of a surfactant-associated protein D characterized by amino acid residues 1-105 of a mouse SPD or the corresponding residues in a human SPD, a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, a mRNA encoding a second region of a surfactant-associated protein D characterized by amino acid residues 106- 256 of a mouse SPD or the corresponding residues in a human SPD, and a mRNA encoding a region of a soluble CD40 ligand characterized by amino acid residues 47-260 of a mouse sCD40L or the corresponding residues in a human sCD40L.

[0077] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a Tissue-type Plasminogen Activator protein, a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, a mRNA encoding a region of a surfactant- associated protein D, and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a Tissue-type Plasminogen Activator protein characterized by amino acid residues 1-22, a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, a mRNA encoding a region of a surfactant-associated protein D characterized by amino acid residues 106-256 of a mouse SPD or the corresponding residues in a human SPD, and a mRNA encoding a region of a soluble CD40 ligand characterized by amino acid residues 47-260 of a mouse sCD40L or the corresponding residues in a human sCD40L.

[0078] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a Tissue-type Plasminogen Activator protein, a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, a mRNA encoding a 2A self-cleavage peptide, a mRNA encoding a region of a surfactant-associated protein D, and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a Tissue-type Plasminogen Activator protein characterized by amino acid residues 1-22, a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, a mRNA encoding a P2A self-cleavage peptide, a mRNA encoding a region of a surfactant-associated protein D characterized by amino acid residues 1-256 of a mouse SPD or the corresponding residues in a human SPD, and a mRNA encoding a region of a soluble CD40 ligand characterized by amino acid residues 47-260 of a mouse sCD40L or the corresponding residues in a human sCD40L.

[0079] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a Tissue-type Plasminogen Activator, a mRNA encoding a SARS- CoV-2 RNA-dependent RNA polymerase, and a mRNA encoding a region of a soluble CD40 ligand. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a Tissue-type Plasminogen Activator characterized by amino acid residues 1-22, a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, and a mRNA encoding a region of a soluble CD40 ligand characterized by amino acid residues 47-260 of a mouse sCD40L or the corresponding residues in a human sCD40L.

[0080] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a Tissue-type Plasminogen Activator and a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase. In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a Tissue-type Plasminogen Activator characterized by amino acid residues 1-22 and a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase.

[0081] In some embodiments, the fusion polynucleotide of this disclosure comprises in order a mRNA encoding a Tissue-type Plasminogen Activator, a mRNA encoding a SARS- CoV-2 RNA-dependent RNA polymerase, a mRNA encoding a 2A self-cleavage peptide, and a mRNA encoding an Interferon-a protein. In some embodiments, the fusion polynucleotide of this disclosure comprises in sequence a mRNA encoding a Tissue-type Plasminogen Activator characterized by amino acid residues 1-22, a mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase, a mRNA encoding a P2A self-cleavage peptide, and a mRNA encoding an Interferon-a protein.

[0082] In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 47. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 48. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 49. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 50. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 52. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 53. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 55. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 56. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 57. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 58. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 59. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 60. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 61. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 63. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 64. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 65. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 66. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 67. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 68. In some embodiments, the fusion polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 69. In some embodiments, the polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 51. In some embodiments, the polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 54. In some embodiments, the polynucleotide of this disclosure comprises the nucleic acid sequence set forth in SEQ ID NO: 62. Lipid Nanoparticles

[0083] RNA, including mRNA, encapsulated lipid nanoparticles (LNPs) mimic low density lipoproteins (LDL) and are taken up by most cell types through receptor-mediated endocytosis. Once in the endosome, ionizable lipids in the RNA encapsulated LNPs respond to the low pH of the endosomes and become cationic. The cationic lipids in the RNA encapsulated LNPs interact with anionic lipids in the endosome and disrupt the endosomal membrane and release the mRNA into the cytoplasm for further processing and translation into the protein that the mRNA encodes.

[0084] In some embodiments, the lipid nanoparticle of this disclosure comprises a RNA fusion polynucleotide as described in any one or more of paragraphs [0042]-[0081],

[0085] In some embodiments, the lipid nanoparticles of this disclosure comprise one or more lipids. In some embodiments, the lipid nanoparticles of this disclosure comprise one or more lipids, wherein the one or more lipids comprises a condensing lipid, a coating lipid, a structural lipid, or combinations thereof. In some embodiments, the one or more lipids of this disclosure comprise a condensing lipid. In some embodiments, the one or more lipids of this disclosure comprise a coating lipid. In some embodiments, the one or more lipids of this disclosure comprise a structural lipid.

[0086] In some embodiments, the structural lipid of this disclosure is selected from the group consisting of hydrogenated soy phosphatidylcholine, fully hydrogenated soy phosphatidylcholine (HSPC), l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), and cholesterol. In some embodiments, the structural lipid of this disclosure is hydrogenated soy phosphatidylcholine. In some embodiments, the structural lipid of this disclosure is fully hydrogenated soy phosphatidylcholine (HSPC). In some embodiments, the structural lipid of this disclosure is l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC). In some embodiments, the structural lipid of this disclosure is cholesterol.

[0087] In some embodiments, the coating lipid of this disclosure is a PEGylated lipid or a poly(2-oxazoline)-conjugated lipid. In some embodiments, the coating lipid of this disclosure is a PEGylated lipid. In some embodiments, the coating lipid of this disclosure is a poly(2-oxazoline)-conjugated lipid. In some embodiments, the PEGylated lipid is selected from the group consisting of l,2-dimyristoylrac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) and 1 ,2-distearoyl-sn-glycero-3 -phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG2000). In some embodiments, the PEGylated lipid is 1, 2-dimyristoylrac-glycero-3 -methoxypolyethylene gly col-2000 (DMG-PEG2000). In some embodiments, the PEGylated lipid is l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG2000).

[0088] In some embodiments, the condensing lipid of this disclosure is a cationic lipid or an ionizable lipid. In some embodiments, the condensing lipid of this disclosure is a cationic lipid. In some embodiments, the condensing lipid of this disclosure is an ionizable lipid. In some embodiments, the cationic lipid is selected from the group consisting of l,2-dioleoyl-3- trimethyl ammoniumpropane, l,2-dioleoyl-3 -trimethylammoniumpropane (chloride salt) (DOTAP), dimethyldioctadecylammonium, and dimethyldioctadecylammonium (bromide salt). In some embodiments, the cationic lipid is l,2-dioleoyl-3 -trimethylammoniumpropane. In some embodiments, the cationic lipid is l,2-dioleoyl-3 -trimethylammoniumpropane (chloride salt) (DOTAP). In some embodiments, the cationic lipid is dimethyldioctadecylammonium. In some embodiments, the cationic lipid is dimethyldioctadecylammonium (bromide salt). In some embodiments, the ionizable lipid is heptatriaconta-6, 9, 28, 3 l-tetraen-19-yl 4-(dimethylamino) butanoate (Dlin-MC3-DMA or MC3).

[0089] In some embodiments, the fusion polynucleotide of this disclosure is encapsulated in a lipid nanoparticle. In some embodiments, the lipid nanoparticle is characterized by lipid nanoparticle size, polydispersity index (PDI), zeta potential, and mRNA encapsulation efficiency.

[0090] In some embodiments, the lipid nanoparticle size of this disclosure is less than 200 nm. In some embodiments, the lipid nanoparticle size of this disclosure is less than 190 nm. In some embodiments, the lipid nanoparticle size of this disclosure is less than 180 nm. In some embodiments, the lipid nanoparticle size of this disclosure is less than 170 nm. In some embodiments, the lipid nanoparticle size of this disclosure is less than 160 nm. In some embodiments, the lipid nanoparticle size of this disclosure is less than 150 nm. In some embodiments, the lipid nanoparticle size of this disclosure is less than 140 nm. In some embodiments, the lipid nanoparticle size of this disclosure is less than 130 nm. In some embodiments, the lipid nanoparticle size of this disclosure is less than 120 nm. In some embodiments, the lipid nanoparticle size of this disclosure is less than 110 nm. In some embodiments, the lipid nanoparticle size of this disclosure is less than 100 nm.

[0091] In some embodiments, the poly dispersity index is less than 0.2.

[0092] In some embodiments, the mRNA encapsulation efficiency of the lipid nanoparticle of this disclosure is greater than 70%. In some embodiments, the mRNA encapsulation efficiency of the lipid nanoparticle of this disclosure is greater than 75%. In some embodiments, the mRNA encapsulation efficiency of the lipid nanoparticle of this disclosure is greater than 80%. In some embodiments, the mRNA encapsulation efficiency of the lipid nanoparticle of this disclosure is greater than 85%. In some embodiments, the mRNA encapsulation efficiency of the lipid nanoparticle of this disclosure is greater than 90%. In some embodiments, the mRNA encapsulation efficiency of the lipid nanoparticle of this disclosure is 90%. In some embodiments, the mRNA encapsulation efficiency of the lipid nanoparticle of this disclosure is 91%. In some embodiments, the mRNA encapsulation efficiency of the lipid nanoparticle of this disclosure is 92%. In some embodiments, the mRNA encapsulation efficiency of the lipid nanoparticle of this disclosure is 93%. In some embodiments, the mRNA encapsulation efficiency of the lipid nanoparticle of this disclosure is 94%. In some embodiments, the mRNA encapsulation efficiency of the lipid nanoparticle of this disclosure is 95%. In some embodiments, the mRNA encapsulation efficiency of the lipid nanoparticle of this disclosure is 96%. In some embodiments, the mRNA encapsulation efficiency of the lipid nanoparticle of this disclosure is 97%. In some embodiments, the mRNA encapsulation efficiency of the lipid nanoparticle of this disclosure is 98%. In some embodiments, the mRNA encapsulation efficiency of the lipid nanoparticle of this disclosure is 99%.

[0093] In some embodiments, the lipid nanoparticle of this disclosure encapsulates between 0.3 mg to 0.6 mg mRNA. In some embodiments, the lipid nanoparticle of this disclosure encapsulates between 0.4 mg to 0.5 mg mRNA. In some embodiments, the lipid nanoparticle of this disclosure encapsulates 0.4 mg mRNA. In some embodiments, the lipid nanoparticle of this disclosure encapsulates 0.41 mg mRNA. In some embodiments, the lipid nanoparticle of this disclosure encapsulates 0.42 mg mRNA. In some embodiments, the lipid nanoparticle of this disclosure encapsulates 0.43 mg mRNA. In some embodiments, the lipid nanoparticle of this disclosure encapsulates 0.44 mg mRNA. In some embodiments, the lipid nanoparticle of this disclosure encapsulates 0.45 mg mRNA. In some embodiments, the lipid nanoparticle of this disclosure encapsulates 0.46 mg mRNA. In some embodiments, the lipid nanoparticle of this disclosure encapsulates 0.47 mg mRNA. In some embodiments, the lipid nanoparticle of this disclosure encapsulates 0.48 mg mRNA. In some embodiments, the lipid nanoparticle of this disclosure encapsulates 0.49 mg mRNA. In some embodiments, the lipid nanoparticle of this disclosure encapsulates 0.5 mg mRNA.

[0094] In some embodiments, the zeta potential of the lipid nanoparticle of this disclosure is between -2 mV to 0 mV. In some embodiments, the zeta potential of the lipid nanoparticle of this disclosure is -2 mV. In some embodiments, the zeta potential of the lipid nanoparticle of this disclosure is -1 mV. In some embodiments, the zeta potential of the lipid nanoparticle of this disclosure is 0 mV.

[0095] In some embodiments, the lipid nanoparticle yield is greater than 40%. In some embodiments, the lipid nanoparticle yield is greater than 50%. In some embodiments, the lipid nanoparticle yield is greater than 60%.

Pharmaceutical Composition

[0096] In some embodiments, a pharmaceutical composition of this disclosure comprises a lipid nanoparticle of this disclosure (i.e. one or more of the lipid nanoparticles of paragraphs [0082]-[0094]) and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is an aqueous solution. In some embodiments, the pharmaceutical composition of this disclosure comprises one or more excipients. In some embodiments, the one or more excipients are selected from the group consisting of a buffer and a pH adjusting agent.

[0097] In some embodiments, the buffer is selected from the group consisting of citrate, sodium citrate tribasic dehydrate, phosphate-buffered saline, and tris (hydroxymethyl) aminomethane (TRIS-base).

[0098] In some embodiments, the pH adjusting agent is citric acid. In some embodiments, the pH of the pharmaceutical composition is between pH 7.0 to pH 7.4.

Methods for Treating or the Manufacture of a Medicament

[0099] In some embodiments, the method for treating or preventing a SARS-CoV-2 infection of this disclosure (i.e. one or more of the lipid nanoparticles of paragraphs [0082]- [0094])comprises administering an effective amount of a lipid nanoparticle of this disclosure to a subject in need or at risk thereof. In some embodiments, the method for treating or preventing a SARS-CoV-2 infection of this disclosure (i.e. one or more of the lipid nanoparticles of paragraphs [0082]-[0094])comprises administering the pharmaceutical composition of this disclosure to a subject in need or at risk thereof.

[0100] In some embodiments, an effective amount of a lipid nanoparticle of this disclosure is used for treating or preventing a SARS-CoV-2 infection in a subject in need or at risk thereof. In some embodiments, a pharmaceutical composition of this disclosure is used for treating or preventing a SARS-CoV-2 infection in a subject in need or at risk thereof.

[0101] In some embodiments, an effective amount of a lipid nanoparticle of this disclosure is used in the manufacture of a medicament for treating or preventing a SARS-CoV-2 infection in a subject in need or at risk thereof. In some embodiments, a pharmaceutical composition of this disclosure is used in the manufacture of a medicament for treating or preventing a SARS-CoV-2 infection in a subject in need or at risk thereof.

[0102] In some embodiments, the methods and uses of this disclosure comprise administering the lipid nanoparticle of this disclosure intravenously, intramuscularly, subcutaneously, or intraperitoneally. In some embodiments, the methods and uses of this disclosure comprise administering the lipid nanoparticle of this disclosure intravenously. In some embodiments, the methods and uses of this disclosure comprise administering the lipid nanoparticle of this disclosure intramuscularly. In some embodiments, the methods and uses of this disclosure comprise administering the lipid nanoparticle of this disclosure subcutaneously. In some embodiments, the methods and uses of this disclosure comprise administering the lipid nanoparticle of this disclosure intraperitoneally.

[0103] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0104] In some embodiments of the methods and uses for treating of this disclosure, an adaptive immune response is stimulated in the treated subject. In some embodiments, the adaptive immune response of this disclosure is characterized by Thl T cell and CD8 T cell activation, i.e., a cellular immune response. In some embodiments, the adaptive immune response of this disclosure is characterized by a CD8+ T cell IFN-y response. In some embodiments, the adaptive immune response of this disclosure is characterized by CD8+ T cell secreting IFN-y. In some embodiments, the adaptive immune response of this disclosure is characterized by little or no antibody production.

[0105] In other embodiments of the methods of treating of this disclosure, an adaptive immune response characterized by both antibodies (a humoral response) and by Thl cell and CD8 T cell activation (a cellular immune response) is observed.

EXAMPLES

Example 1. mRNA Synthesis and Purification

[0106] To enzymatically synthesize mRNA, linearized plasmid DNA templates were first designed to contain a gene of interest (e.g., SARS-CoV-2 spike protein or region, surfactant- associated protein D, soluble CD40 ligand, and 2A domain) T7 RNA polymerase promoter sequence, a 5’-UTR, an open reading frame (ORF), and 3’-UTR. A poly(A) tail was also added into the DNA template via PCR (an alternative would have been to post- transcriptionally add the poly(A)tail by enzymatic polyadenylation). The DNA template also has a unique restriction enzyme site for linearization downstream of the desired 3 ’-end. Following restriction digest (or PCR), the DNA template was purified using standard DNA purification procedures or kits to remove enzymes and reaction components.

[0107] In vitro transcription (IVT) with CleanCap® AG of the DNA template was performed to prepare a crude mRNA extract. The co-transcriptional CleanCap® AG analog was used to produce a cap 1 mRNA. The components of the reaction were prepared in the order listed in Table 1 below. After the addition of lOx transcription buffer, the mixture was vigorously mixed (e.g., vortex) prior to the addition of the DNA template. After all IVT components were added and mixed, the reaction was incubated at 37°C in a water bath or heat block for 2-3 hours. The IVT reaction mixture was optionally treated with DNase I to remove any contaminating DNA in the crude mRNA extract.

[0108] mRNA was purified using a lithium chloride precipitation procedure. The crude mRNA extract was mixed with lithium chloride (2.5 M final concentration), then chilled at - 20°C for at least 30 minutes to form a precipitate. The mixture was centrifuged for 30 minutes at 18,500 x g, 4°C, then the supernatant was discarded and the pellet retained. The pellet was washed with cold 70% ethanol, then centrifuged for 5 minutes at 12,000 x g, 4°C. The ethanol supernatant was discarded, then the ethanol wash was repeated. The pellet was dried and resuspended in buffer (e.g., 1 mM EDTA or 1 mM sodium citrate at pH 6-8).

Table 1. In Vitro Transcription Components

Example 2. Lipid Nanoparticle Formulation

[0109] mRNA encapsulated lipid nanoparticles (LNPs) were prepared by mixing an ethanolic solution of lipids (e.g., GenVoy-ILM™) with an aqueous solution e.g., PNI Formulation Buffer) of RNA at a low pH (e.g., pH 4). The formulations were manufactured using NanoAssemblr® Ignite™ equipped with NIN002 NxGen™ mixers. After mixing, the change in polarity of the environment triggered self-assembly of the LNPs. The low pH caused the ionizable lipids to become cationic, where they first interact with anioic RNA through electrostatic complexation to form the particle core. Other lipids (e.g., cholesterol, structural lipids, coating lipids, DSPC, and Peg stabilizer) assembled around the core. Rapid mixing allowed for homogenous conditions and promoted core formation overgrowth, leading to a homogenous population of LNPs. The formulated LNPs from the instrument were processed for ethanol removal, buffer exchange, adjusted to a concentration of interest, and sterile filtered (e.g., 0.2 pm pore size or 0.45 pm pore size). The adjusted mRNA concentration, LNP size based on the diameter (d. [nm]), poly dispersity index (PDI), zeta potential (ZP), and LNP yield were measured (Table 2). The mRNA encapsulated LNPs are neutral at physiological pH to eliminate toxicity.

[0110] Zeta potential (ZP) was measured to determine the charge on the surface of the LNPs in milli Volts (mV). PDI was measured by DLS to determine the uniformity of particle size of the nanoparticulate dispersion. Encapsulation efficiency (EE) was determined by the N/P ratio, which is the molar ratio between amines (N, which became cationic at low pH) found on the ionizable lipids, and the phosphates (P, anionic) found on the RNA backbone.

Table 2. mRNA Encapsulated Lipid Nanoparticle (LNP) Formulations

Example 3. Response to SARS-CoV-2 mRNA vaccine

[0111] A study to determine the response to a SARS-CoV-2 mRNA vaccine or control constructs was performed with seventy mice that were separated into seven groups of ten mice each group (Table 3). Mice were intramuscularly injected in the leg with a vehicle control or mRNA encapsulated in lipid nanoparticles (e.g., LNP-1, LNP -2, LNP-3, LNP-4, LNP-5, or LNP-6) on study day (SD) 0 and SD 28. Blood was collected in-life on SD 0 (pretreatment), SD 14 and SD 28 (before the second injection). The weight of the mice was measured weekly to observe body weight change (Figure 4). Observations were taken on days of procedures and once daily for 7 days after SD 28 treatment. Observations were also taken once daily for 2 days after SD14 in-life blood collection. Terminal collections for blood and spleens were performed on SD 49. Table 3. Study Design

[0112] A standard indirect ELISA (e.g., IgG ELISA) was performed to analyze the blood samples from the mice in the study for antibodies that bound to the SARS-CoV-2 spike protein. Nunc™ MaxiSorp™ 96-well plates were coated with 100 pL of SARS-CoV-2 spike protein (Sino Biological) diluted to 2 pg/mL in IX PBS, pH 7.4. The plates were incubated statically for 12 hours at 37°C. Unbound coating antigen in each well was removed by washing 3 times with 100 pL IX PBS + 0.05% Tween-20. The plates were blocked in IX PBS + 5% skim milk for 1 hr. at 37°C. Test and positive control samples were diluted in assay diluent (IX PBS + Tween-20 + 1% skim milk) to a 1 :20 starting point dilution followed by five-folds serial dilution using U-bottom dilution plates. Once blocking was completed, the blocking buffer was removed by inversion and each sample was plated in triplicates. Plates were incubated for 2 hours at 37°C statically, followed by washing 3x times with 100 pL of IX PBS-Tween-20 to remove unbound sera. Secondary detection antibody (goat anti-species-HRP IgG, Abeam) was diluted to 1 : 10,000 and 100 pL was added to each well. The plates were incubated for 30 minutes at room temperature statically, and unbound antibodies were subsequently removed by washing 3x times with 100 pL of IX PBS-Tween-20. To develop the plate, 100 pL of 1-Step™ Ultra tetramethylbenzidine (TMB) substrate was added to each well. The reaction was stopped after about 10 minutes with 50 pL of TMB stop solution (SeraCare®). The plates were read within 30 minutes at 450 nm with a Thermo Labsystems Multiskan™ spectrophotometer. Titers were defined as reciprocal of the dilutions that generated a specific cut-off value at an optical density of 0.3 at 450 nm (OD450) on the linear part of the titration curve. The LNP-1 titer was comparable to the diluent control, while the other LNP groups showed an increasing trend over time posttreatment (Figure 5).

[0113] An enzyme-linked immunospot (ELISpot) assay was performed to quantify the number of cytokine-secreting cells. Cytokines secreted include, but are not limited to, Interferon-y (IFN-y) and Interleukin-4 (IL-4). The spleens were collected from each mouse in the study on the day of termination (SD 49) at necropsy. The spleens were harvested and collected in tubes containing chilled RPMI medium on wet ice until it was processed for the downstream assays. Prior to processing, the spleens were placed into a sterile petri dish containing medium. The back of the plunger of the lOcc syringe was used to homogenize the spleens. The homogenate was passed through a filter and transferred into a sterile tube and placed on ice. The homogenate was then centrifuged at 1200 rpm for 8 minutes. The supernatant was gently poured off, and the edge of the tube was blotted with a clean paper towel. Ammonium-chloride-potassium (ACK) lysis buffer was added to lyse the red blood cells, and the cells incubated at room temperature for about 3 minutes. The cells were then filtered through a 100 pm filter and centrifuged at 1200 rpm for 8 minutes. The supernatant was poured off, and the cells were resuspended in 2 mM L-Glutamine CTL-Test™ Medium. The suspension was filtered into a new 15 mL conical tube. The cells were maintained on ice until assayed.

[0114] A dual color IFN-y /IL-4 ELISpot assay was performed using CTL ELISpot kits. All solutions were freshly made prior to use. On Day 1, the CTL-Test™ Media was prepared. The plates were coated with anti-IFN- y and anti-IL-4 and incubated overnight at 4 °C. The antigen/mitogen solutions were prepared at two times the final concentration in the medium. The final concentrations of the solutions include: 2 pg/mL SARS-CoV-2 spike-specific peptide pool (PepMix™ Pool 158+157) and 1 pg/mL Concanavalin A (Con A). 100 pL/well antigen/mitogen solutions were plated. The plate containing the antigens were placed into a 37 °C incubator for 10-20 minutes before plating cells. The cells were adjusted to a concentration of 3 x 10⁶ cells/mL in the medium (z.e., 0.3 x 10⁵ splenocytes/well). The cells were then incubated at 37°C in a humidified incubator, 5-9% CO2, until plated.

[0115] On Day 2, the buffer solutions were prepared: IX PBS, distilled water and 0.05% Tween-PBS. The anti-murine IFN-y and anti- murine IL-4 detection solution was prepared. The plate was washed two times with IX PBS and then two times with 0.05% Tween-PBS, 200 pL/well each time. A volume of 80 pL/well anti-murine IFN-y and anti-murine IL-4 detection solution was added, then the plate was incubated at room temperature for two hours. The tertiary solution was then prepared. The plates were washed three times with 0.05% Tween-PBS, 200 pL/well, and then the tertiary solution was added at 80 pL/well. The plate was incubated at room temperature for 30 minutes. The plate was washed two times with 0.05% Tween-PBS, and then two times with distilled water, 200 pL/well each time. The blue developer solution was added for IL-4 detection at 80 pL/well, and the plates incubated at room temperature for 15 minutes. The reaction was stopped by gently rinsing the membrane in the plate with tap water, decanting the solution, and repeating this three times. The red developer solution was added for IFN-y detection at 80 pL/well, and the plates incubated at room temperature for 15 minutes. The reaction was stopped by gently rinsing the membrane with tap water, decanting the solution, and repeating this three times. The plates were air-dried overnight in running laminar flow hood. The plates were scanned (CTL) and analyzed using the CTL ImmunoSpot® Analyzer and ImmunoSpot® Software (Cellular Technology). Splenocytes treated with the peptide pool that secreted IFN-y were detectable in all the groups except LNP-6, as compared to the diluent control (Figure 6A). Splenocytes treated with the peptide pool that secreted IL-4 were detectable in all groups as compared to the diluent control (Figure 7A).

[0116] A Plaque Reduction Neutralization Test (PRNT assay) was conducted to determine which mRNA encapsulated LNP enhances activation of T cell helper function by measuring the ability of serum antibodies to neutralize a virus. The PRNT assay was performed using serum samples from SD 49. First, Vero E6 cells (ATCC, Catalog No. CRL-1586) were plated in 24-well plates at 175,000 cells/well in DMEM + 10% FBS + Gentamicin. The plates were incubated at 37°C, 5.0% CO2, until the cells reached 80%-100% confluency the following day. On the assay day, the serum samples were heat inactivated at 56°C for 30 minutes. The assay set-up was performed as follows: in a 96-well deep well plate, 405 pL of diluent (DMEM + 2% FBS + gentamicin) was added to column 1 and 300 pL of diluent was added to columns 3, 5, 7, 9 and 11. Forty-five (45) pL of the heat-inactivated serum sample was added into the first column (1 : 10 dilution). When all samples were added, the contents of the wells were mixed up and down about 5 times, and 150 pl was transferred from column 1 to column 3 for a 1 :3-fold dilution. This was repeated for the next set of wells down the plate (i.e., from column 5 to column 7 and from column 9 to column 11) and 150 pl was discarded from this last dilution for an identical final volume in all wells. For the virus positive control (e.g., rabbit reference serum), 300 pL of diluent was added to columns 1, 3, 5, 7, 9 and 11 while 600 pL of diluent was added to 1 row, representing the negative control.

[0117] A 30 pfu/well concentration of the Washington State strain WA/2020 virus was prepared and kept on ice until use. After the 96-well titration plate was prepared as described above, 300 pL of 30 pfu/well virus dilution was added to all samples and positive control wells. This doubled the sample dilution factor (first well begins at 1 :20 dilution). The plate was then covered with a plate sealer and incubated at 37°C, 5.0% CO2 for 1 hour. After incubation, the media from the 24-well plate with Vero E6 cells was removed and 250 pL of titrated samples was added in duplicate from the titration plate. Only one plate was prepared at a time to avoid drying out the cells. The 24-well plates were incubated at 37°C, 5.0% CO2 for 1 hour for viral infection. During this time, 0.5% methylcellulose media was heated in a 37°C water bath. After one-hour incubation, 1 mL of the 0.5% methylcellulose media was added to each well and the plates were incubated at 37°C, 5% CO2 for 3 days. The methylcellulose medium was removed, and the plates were washed once with 1 mL IX PBS. The plates were fixed with 400 pL ice cold methanol per well at -20°C for 30 minutes. After fixation, the methanol was discarded, and the monolayers were stained with 250 pL per well of 0.2% crystal violet (20% MeOH, 80% dFLO) for 30 minutes at room temperature. The plates were finally washed once with dFLO and were left to dry for at least 15 minutes. The plaques in each well were recorded and the IC50 titer (Figure 8) and IC90 titer were calculated based on the average number of plaques detected in the virus control wells. A control (rabbit) reference serum with established titer (5,400 IC50) was included in each assay set-up to serve as an internal positive control. T cell helper function was activated in the LNP-2, LNP-3, LNP-4 and LNP-5 groups as compared to the diluent control (Figure 8). The LNP-3 showed greater neutralization activity than LNP-2, LNP-4, and LNP-5. LNP-3 leads to separate protein expression of full-length SARS-CoV-2 Spike protein and multimerized soluble CD40 ligand (sCD40L) and multimerized surfactant-associated protein D (SPD) (Figure 1C, Panel h and Figure 11), while LNP-2 leads to protein expression of a truncated SARS-CoV-2 spike protein fused to multimerized sCD40L and SPD (Figure 1 A, Panel b and Figure 9). LNP-4 leads to protein expression of truncated SARS-CoV-2 spike protein fused to multimerized sCD40L (Figure 1 A, Panel c and Figure 9), while LNP-5 leads to expression of full-length SARS-CoV-2 spike protein (Figure IB, Panel f and Figure 9).

[0118] Table 4 summarizes the antibody, Thl and Th2 responses in mice and endosomal trapping capability after treatment with various LNP mRNA vaccines.

Table 4. Cellular and Immunological Response to LNP mRNA vaccine

Exemplary Constructs

[0119] Non-limiting examples of the fusion polynucleotides and encoded proteins of this disclosure comprise one or more of the regions, domains or full-length sequences of Table 5 and the fusion polynucleotides and the encoded proteins of this disclosure are shown in Table 6

Table 5.

Table 6. Fusion polynucleotides and the encoded proteins

Ill

Claims

CLAIMS We claim:

2. The fusion polynucleotide according to claim 1, wherein the fusion polynucleotide further comprises a mRNA encoding a 2A self-cleaving peptide.

5. The fusion polynucleotide according to claim 4, wherein the cell-bound CD40 ligand is characterized by the amino acid sequence of the full-length protein.

6. The fusion polynucleotide according to claim 4, wherein the mRNA encoding a cell-bound CD40 ligand has a deletion of one or more nucleotides.

7. The fusion polynucleotide according to claim 5, wherein the cell-bound CD40 ligand has a deletion in the amino acid sequence of the full-length protein extending from amino acid residue 110 to amino acid residue 122.

11. The fusion polynucleotide according to claim 10, wherein the fusion polynucleotide further comprises a mRNA encoding an Interferon-a protein.

12. The fusion polynucleotide according to claim 10, wherein the fusion polynucleotide further comprises a mRNA encoding a region of a soluble CD40 ligand.

13. The fusion polynucleotide according to claim 10 or 12, wherein the fusion polynucleotide further comprises a mRNA encoding one or more regions of a surfactant-associated protein D.

14. The fusion polynucleotide according to any one of claims 11 or 13, wherein the fusion polynucleotide further comprises a mRNA encoding a 2A self-cleaving peptide.

15. The fusion polynucleotide according to any one of claims 1, 9, 13 and 14, wherein one of the one or more regions of the surfactant-associated protein D is an N-terminal region of the surfactant-associated protein D.

16. The fusion polynucleotide according to claim 1, 9, 13 and 14, wherein one of one or more regions of the surfactant-associated protein D is 50 amino acids to 300 amino acids in length.

17. The fusion polynucleotide according to claim 16, wherein one of the one or more regions of the surfactant-associated protein D is 80 amino acids to 260 amino acids in length.

18. The fusion polynucleotide according to claim 17, wherein one of the one or more regions of the surfactant-associated protein D is 95 amino acids to 160 amino acids in length.

19. The fusion polynucleotide according to claim 18, wherein one of the one or more regions of the surfactant-associated protein D is 105 amino acids in length.

20. The fusion polynucleotide according to claim 18, wherein one of the one or more regions of the surfactant-associated protein D is 151 amino acids in length.

21. The fusion polynucleotide according to claim 17, wherein one of the one or more regions of the surfactant-associated protein D is 256 amino acids in length.

22. The fusion polynucleotide according to any one of claims 23-29, wherein the one of the one or more regions of the surfactant-associated protein D is located at the N-terminus of the region of SARS-CoV-2 spike protein.

23. The fusion polynucleotide according to any one of claims 15-22, wherein one of the one or more regions of the surfactant-associated protein D is located at the C-terminus of the region of SARS-CoV-2 spike protein.

24. The fusion polynucleotide according to any one of claims 15-21, wherein one of the one or more regions of the surfactant-associated protein D is located at the N-terminus of the SARS-CoV-2 RNA-dependent RNA polymerase.

25. The fusion polynucleotide according to any one of claims 15-21 and 24, wherein one of the one or more regions of the surfactant-associated protein D is located at the C-terminus of the SARS-CoV-2 RNA-dependent RNA polymerase.

26. The fusion polynucleotide according to claim 23 or 25, wherein one of the one or more regions of the surfactant-associated protein D is located at the C-terminus of the 2A selfcleaving peptide.

27. The fusion polynucleotide according to any one of claims 1-3, 9, 15-23 and 26, wherein the region of the soluble CD40 ligand is located at the C-terminus of the region of the SARS- CoV-2 spike protein.

28. The fusion polynucleotide according to claim 1-3, 9 and 13-23, 26 and 27, wherein the region of the soluble CD40 ligand is located at the C-terminus of one of the one or more regions of the surfactant-associated protein D.

29. The fusion polynucleotide according to any one of claims 9 and 12-21, and 24-26, wherein the soluble CD40 ligand is located at the C-terminus of the region of the SARS- CoV-2 RNA-dependent RNA polymerase.

30. The fusion polynucleotide according to any one of claims 9 and 12-29, wherein the soluble CD40 ligand is located at the C-terminus of the region of the one of the one or more region of the surfactant-associated protein D.

31. The fusion polynucleotide according to any one of claims 1-3, 9 and 12-30, wherein the region of soluble CD40 ligand is 200 to 230 amino acids in length.

32. The fusion polynucleotide according to claim 31, wherein the region of the soluble CD40 ligand is 214 amino acids in length.

33. The fusion polynucleotide according to any one of claims 4-7, wherein the cell-bound CD40 ligand is located at the C-terminus of the region of the SARS-CoV-2 spike protein.

34. The fusion polynucleotide according to any one of claims 4-7, wherein the cell-bound CD40 ligand is located at the C-terminus of the 2A self-cleaving peptide.

35. The fusion polynucleotide according to claim 33 or 34, wherein the cell-bound CD40 ligand is 230 to 260 amino acids in length.

36. The fusion polynucleotide according to claim 35, wherein the cell-bound CD40 ligand is 260 amino acids in length.

37. The fusion polynucleotide according to claim 35, wherein the cell-bound CD40 ligand is 247 amino acids in length.

38. The fusion polynucleotide according to claim 8, wherein the Interferon-a protein is located at the C-terminus of the SARS-CoV-2 spike protein.

39. The fusion polynucleotide according to claim 11, wherein the Interferon-a protein is located at the C-terminus of the SARS-CoV-2 RNA-dependent RNA polymerase.

40. The fusion polynucleotide according to any one of claims 8, 14, 38 or 39, wherein the Interferon-a protein is located at the C-terminus of the 2A self-cleaving peptide.

41. The fusion polynucleotide according to any one of claims 38-40, wherein the Interferon-a protein is 189 to 190 amino acids in length.

42. The fusion polynucleotide according to claim 41, wherein the Interferon-a protein is 190 amino acids in length.

43. The fusion polynucleotide according to any one of claims 2, 4-8, and 14-42, wherein the 2A self-cleaving peptide is 18 to 23 amino acids in length.

44. The fusion polynucleotide according to claim 43, wherein the 2A self-cleaving peptide is 22 amino acids in length.

45. The fusion polynucleotide according to any one of claims 10-21, 24-26, 29-32, and 39-44, wherein the Tissue-type Plasminogen Activator protein is an N-terminal region of that protein.

46. The fusion polynucleotide according to claim 10-21, 24-26, 29-32, and 39-45, wherein the Tissue-type Plasminogen Activator protein is located at the N-terminus of the SARS- CoV-2 RNA-dependent RNA polymerase.

47. The fusion polynucleotide according to any one of claims 1-8, 15-23, 26-28, 31-38, and 40-44, wherein the region of the SARS-CoV-2 spike protein is 1,100 amino acids to 1,300 amino acids in length.

48. The fusion polynucleotide according to claim 47, wherein the region of the SARS-CoV-2 spike protein is 1,192 amino acids in length.

49. The fusion polynucleotide according to claim 47, wherein the region of the SARS-CoV-2 spike protein is 1,208 amino acids in length.

50. The fusion polynucleotide according to claim 47, wherein the region of the SARS-CoV-2 spike protein is 1,273 amino acids in length.

51. The fusion polynucleotide according to any one of claims 1-50, wherein the fusion polynucleotide is encapsulated in the lipid nanoparticle.

52. The fusion polynucleotide according to any one of claims 1-8, 15-23, 26-28, 30-38, 40-

44, and 47-51, wherein the fusion polynucleotide encodes a soluble SARS-CoV-2 spike protein.

53. The fusion polynucleotide according to any one of claims 1-8, 15-23, 26-28, 30-38, 40-

44, and 47-51, wherein the fusion polynucleotide encodes a cell-bound SARS-CoV-2 spike protein .

54. The fusion polynucleotide according to any one of claims 9-21, 24-26, 29-32, 39-46 and

51, wherein the fusion polynucleotide encodes a soluble SARS-CoV-2 RNA-dependent RNA polymerase.

55. The fusion polynucleotide according to any one of claims 9-21, 24-26, 29-32, 39-46 and

51, wherein the fusion polynucleotide encodes a cell-bound SARS-CoV-2 RNA-dependent RNA polymerase.

56. The fusion polynucleotide according to claim 52 or 54, wherein the soluble protein encoded by the fusion polynucleotide is transiently expressed.

57. The fusion polynucleotide according to claim 53 or 55, wherein the cell-bound protein encoded by the fusion polynucleotide is transiently expressed.

58. The fusion polynucleotide according to any one of claims 1-8, 15-23, 26-28, 30-38, 40-

44, 47-53 and 56-57, wherein the mRNA encoding a region of a SARS-CoV-2 spike protein is a modified mRNA.

59. The fusion polynucleotide according to any one of claims 9-21, 24-26, 29-32, 39-46, 51, and 54-57, wherein the mRNA encoding a SARS-CoV-2 RNA-dependent RNA polymerase is a modified mRNA.

60. The fusion polynucleotide according to any one of claims 1, 2, 9, 13-32, and 40-59, wherein the mRNA encoding one or more regions of a surfactant-associated protein D is a modified mRNA.

61. The fusion polynucleotide according to any one of claims 1-3, 9, 12-33, 38, and 40-60, wherein the mRNA encoding a region of a soluble CD40 ligand is a modified mRNA.

62. The fusion polynucleotide according to any one of claims 4-7, 33-38, 43, 44, 47-53, and 56-58, wherein the mRNA encoding the cell-bound CD40 ligand or is a modified mRNA.

63. The fusion polynucleotide according to any one of claims 8, 11-14, and 38-62, wherein the mRNA encoding the Interferon-a protein is a modified mRNA.

64. The fusion polynucleotide according to any one of claims 2, 4-8, and 14-63 , wherein the mRNA encoding the 2A self-cleavage peptide is a modified mRNA.

65. The fusion polynucleotide according to any one of claims 10-14, 28-32, 39-46, 51, 54-57, and 59-64, wherein the Tissue-type Plasminogen Activator protein is a modified mRNA.

66. The fusion polynucleotide according to any one of claims 58-65, wherein the modified mRNA is characterized by one or more uridines modified to pseudouridine.

67. The fusion polynucleotide according to any one of claims 1-8, 15-23, 26-28, 30-38, 40- 44, 47-53, 56-58 and 60-66, wherein a variant of the SARS-CoV-2 spike protein is selected from the group consisting of an alpha variant, a beta variant, a gamma variant, a delta variant, an omicron variant, an omicron BA.1 variant, an omicron B A.2 variant, and a deltacron variant.

47.

48.

49.

50.

52.

53.

55.

56.

76. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

57.

77. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO:

58.

59.

80. A fusion polynucleotide comprising the nucleic acid sequence set forth in SEQ ID NO: 61.

63.

64.

65.

66.

67.

68.

69.

88. A lipid nanoparticle comprising a fusion polypeptide of any one of claims 1-87.

90. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 25.

91. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 26.

100. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 35.

101. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 36.

110. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 45.

111. A lipid nanoparticle comprising a fusion polynucleotide, wherein the fusion polynucleotide encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 46.

112. The lipid nanoparticle according to any one of claims 88-111, wherein the lipid nanoparticle is capable of inducing a Thl immune response on administration to a subject.

113. The lipid nanoparticle according to claim 112, wherein the Thl immune response is a CD8+ T cell stimulated Interferon-gamma (IFN-y) response.

120. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding a 2A self-cleaving peptide, and a mRNA encoding a cell-bound CD40 ligand.

121. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a region of a SARS-CoV-2 spike protein, a mRNA encoding a 2A self-cleaving peptide, and a mRNA encoding a cell-bound CD40 ligand.

128. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a Tissue Plasminogen Activator, a mRNA encoding a SARS-CoV-2 RNA- dependent RNA polymerase, and a mRNA encoding a region of a soluble CD40 ligand.

129. A lipid nanoparticle comprising a fusion polynucleotide comprising in order a mRNA encoding a Tissue Plasminogen Activator and a mRNA encoding a SARS-CoV-2 RNA- dependent RNA polymerase.

131. The lipid nanoparticle according to any one of claims 88-130, wherein the lipid nanoparticle comprises one or more lipids.

132. The lipid nanoparticle according to claim 131, wherein the one or more lipids comprises a condensing lipid, a coating lipid, a structural lipid, or combinations thereof.

133. The lipid nanoparticle according to claim 132, wherein the structural lipid is selected from the group consisting of hydrogenated soy phosphatidylcholine, fully hydrogenated soy phosphatidylcholine (HSPC), l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC), and cholesterol.

134. The lipid nanoparticle according to claim 132, wherein the coating lipid is a PEGylated lipid or a poly(2-oxazoline)-conjugated lipid.

135. The lipid nanoparticle according to claim 134, wherein the PEGylated lipid is selected from the group consisting of l,2-dimyristoylrac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) and 1 ,2-distearoyl-sn-glycero-3 -phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG2000).

136. The lipid nanoparticle according to claim 132, wherein the condensing lipid is a cationic lipid or an ionizable lipid.

137. The lipid nanoparticle according to claim 136, wherein the cationic lipid is selected from the group consisting of l,2-dioleoyl-3 -trimethylammoniumpropane, l,2-dioleoyl-3- trimethyl ammoniumpropane (chloride salt) (DOTAP), dimethyldioctadecylammonium, and dimethyldioctadecylammonium (bromide salt).

138. The lipid nanoparticle according to claim 136, wherein the ionizable lipid is heptatriaconta-6, 9, 28, 3 l-tetraen-19-yl 4-(dimethylamino) butanoate (Dlin-MC3-DMA or MC3).

139. A pharmaceutical composition comprising a fusion polynucleotide according to any one of claims 1-87 or a lipid nanoparticle according to any one of embodiments 88-138 and a pharmaceutically acceptable carrier.

140. The pharmaceutical composition according to claim 139, wherein the pharmaceutically acceptable carrier is an aqueous solution.

141. The pharmaceutical composition according to claim 139 or 140, wherein the pharmaceutical composition comprises one or more excipients.

142. The pharmaceutical composition according to claim 141, wherein the one or more excipients are selected from the group consisting of a buffer and a pH adjusting agent.

143. The pharmaceutical composition according to claim 142, wherein the buffer is selected from the group consisting of citrate, sodium citrate tribasic dehydrate, phosphate-buffered saline, and tris (hydroxymethyl) aminomethane (TRIS-base).

144. The pharmaceutical composition according to claim 142, wherein the pH adjusting agent is citric acid.

145. A method for treating or preventing a SARS-CoV-2 infection comprising administering an effective amount of the lipid nanoparticle of any one of claims 88-138 or the pharmaceutical composition of any one of claims 139-144 to a subject in need or at risk thereof.

146. A method for stimulating an adaptive immune response comprising administering the lipid nanoparticle of any one of claims 88-138 or the pharmaceutical composition of any one of claims 139-144 to a subject in need thereof.

147. The method according to claim 146, wherein the adaptive immune response is characterized by CD8+ T cell activation.

148. The method according to claim 146 or 147, wherein the adaptive immune response is characterized by a CD8+ T cell IFN-y response.

149. A method for stimulating a Thl immune response comprising administering the lipid nanoparticle of any one of embodiments 88-138 or the pharmaceutical composition of any one of claims 139-144 to a subject in need thereof.

150. The method according to claim 149, wherein the Thl immune response is characterized by a lack of IgG production.

151. The method according to claim 149 or 150, wherein the Thl immune response is characterized by a downstream CD8+ T cell induction.

152. The method according to any one of claims 149-151, wherein the Thl immune response is characterized by a downstream CD8+ T cell IFN-y response.

153. A method for stimulating a CD8+ T cell IFN-y response comprising administering the lipid nanoparticle of any one of claims 88-138 or the pharmaceutical composition of any one of claims 139-144 to a subject in need thereof.

154. The method according to any one of embodiments 145-153, wherein the lipid nanoparticle of any one of claims 88-138 or the pharmaceutical composition of any one of claims 139-144 is administered intravenously, intramuscularly, subcutaneously, or intraperitoneally.

155. The method according to any one of claims 145-154, wherein the subject is a mammal.

156. The method according to claim 155, wherein the subject is a human.