JPWO2021198963A5 - - Google Patents

Description

In some embodiments, the VLPs cross-compete with other severe acute respiratory syndrome coronaviruses and/or human betacoronaviruses for binding to the ACE2 receptor, neuropilin-1, and/or other receptors.
Further aspects of the invention are described below:
[Item 1]
an expression cassette comprising a nucleic acid sequence encoding a recombinant protein comprising a conserved amino acid sequence derived from a virus fused to an immunogenic amino acid sequence;
target sequences for a first recombinase flanking said expression cassette;
one or more additional target sequences for one or more additional recombinases integrated within the non-binding regions of the target sequence for the first recombinase;
An expression vector comprising:
An expression vector, wherein a protein expressed from said expression cassette in a cell is capable of forming a virus-like particle (VLP).
[Item 2]
Item 2. The expression vector of Item 1, wherein the immunogenic amino acid sequence is derived from the same virus as the conserved amino acid sequence.
[Item 3]
Item 3. The expression vector according to item 1 or 2, wherein the conserved amino acid sequence is derived from a viral glycoprotein.
[Item 4]
4. The expression vector of claim 3, wherein the immunogenic amino acid sequence is derived from the same viral glycoprotein.
[Item 5]
Item 5. The expression vector according to any one of Items 1 to 4, further comprising a nucleic acid sequence encoding a viral envelope protein and/or a nucleic acid sequence encoding a viral matrix protein.
[Item 6]
Item 6. The expression vector according to Item 5, wherein the viral envelope protein and/or the viral matrix protein is derived from the same virus as the conserved amino acid sequence.
[Item 7]
Item 7. The expression vector according to any one of Items 1 to 6, wherein the conserved amino acid sequence, the immunogenic amino acid sequence, the viral envelope protein, and/or the viral matrix protein are consensus sequences.
[Item 8]
Item 8. The expression vector of any one of items 1 to 7, wherein the recombinant protein is capable of stimulating an immune response against the virus, including neutralizing antibodies.
[Item 9]
Item 9. The expression vector of any one of items 1 to 8, wherein the recombinant protein is capable of stimulating a Th1 cell-mediated immune response against the virus.
[Item 10]
10. The expression vector of claim 8 or 9, wherein the immune response is cross-reactive to related viruses or related strains.
[Item 11]
Item 11. The expression vector of any one of items 1 to 10, wherein the recombinant protein excludes viral-derived amino acid sequences that stimulate immune responses including non-neutralizing antibodies and/or stimulate Th2 cell-mediated immune responses.
[Item 12]
Item 12. The expression vector of any one of items 1 to 11, wherein the expression cassette comprises a single open reading frame comprising a nucleic acid sequence encoding a self-cleaving peptide between each nucleic acid sequence encoding a protein.
[Item 13]
Item 13. The expression vector according to any one of Items 1 to 12, wherein the virus is a coronavirus, an influenza virus, a human immunodeficiency virus, a human papilloma virus, a hepatitis virus, or an oncolytic virus.
[Item 14]
14. The expression vector of claim 13, wherein the virus is a coronavirus.
[Item 15]
Item 15. The expression vector of item 14, wherein the coronavirus is COVID-19.
[Item 16]
16. The expression vector of claim 14 or 15, wherein the expression cassette comprises a nucleic acid sequence encoding a recombinant protein comprising conserved and immunogenic amino acid sequences derived from a coronavirus membrane (M) protein, a coronavirus envelope (E) protein, and a coronavirus spike (S) protein.
[Item 17]
17. The expression vector of claim 16, wherein the conserved amino acid sequence is derived from the S protein S2' cleavage site and internal fusion peptide (IFP).
[Item 18]
18. The expression vector of claim 16 or 17, wherein the conserved amino acid sequence comprises SEQ ID NO:12.
[Item 19]
Item 19. The expression vector of any one of Items 16 to 18, wherein the immunogenic amino acid sequence is derived from the S protein receptor binding domain (RBD).
[Item 20]
20. The expression vector of any one of claims 16 to 19, wherein the immunogenic amino acid sequence is at least about 90% identical to SEQ ID NO:11.
[Item 21]
21. The expression vector of any one of claims 16 to 20, wherein the recombinant protein further comprises a transmembrane (TM) domain sequence derived from the S protein.
[Item 22]
22. The expression vector of any one of claims 16 to 21, wherein the recombinant protein excludes amino acid sequences from the S protein that stimulate immune responses that include non-neutralizing antibodies and/or stimulate Th2 cell-mediated immune responses.
[Item 23]
22. The expression vector of any one of paragraphs 16 to 21, wherein the amino acid sequence of the recombinant protein is at least about 90% identical to SEQ ID NO:55.
[Item 24]
24. The expression vector of any one of claims 16 to 23, wherein the expression cassette comprises a single open reading frame translated as an amino acid sequence that is at least about 90% identical to SEQ ID NO:57.
[Item 25]
25. The expression vector of any one of claims 16 to 24, wherein the recombinant protein is capable of stimulating an immune response against COVID-19.
[Item 26]
26. The expression vector of any one of claims 16 to 25, wherein the recombinant protein is capable of stimulating a Th1 cell-mediated immune response against COVID-19.
[Item 27]
27. The expression vector of claim 25 or 26, wherein the immune response is cross-reactive to other coronaviruses.
[Item 28]
28. The expression vector of claim 27, wherein the immune response is cross-reactive to other severe acute respiratory syndrome coronaviruses and/or human betacoronaviruses.
[Item 29]
29. The expression vector of any one of paragraphs 1 to 28, wherein the target sequence for the first recombinase and the one or more additional target sequences for the one or more additional recombinases are selected from the group consisting of a PY54 pal site, an N15 telRL site, a loxP site, a φK02 telRL site, an FRT site, a phiC31 attP site, and a λ attP site.
[Item 30]
30. The expression vector according to claim 29, comprising each of the target sequences.
[Item 31]
31. The expression vector of claim 30, comprising the Tel recombinase pal site and the telRL, loxP, and FRT recombinase target binding sequences integrated within the pal site.
[Item 32]
Item 32. The expression vector according to any one of Items 1 to 31, which is a vector for producing a vector that does not contain a bacterial sequence.
[Item 33]
33. The expression vector of claim 32, wherein the bacterial sequence-free vector has circular covalently closed ends.
[Item 34]
33. The expression vector of claim 32, wherein the bacterial sequence-free vector has linear, covalently linked closed ends.
[Item 35]
35. The expression vector of any one of paragraphs 1 to 34, further comprising at least one enhancer sequence flanking the target sequence for the first recombinase.
[Item 36]
36. The expression vector of claim 35, wherein the at least one enhancer sequence is at least two enhancer sequences.
[Item 37]
37. The expression vector of claim 35 or 36, wherein at least one enhancer sequence is an SV40 enhancer sequence.
[Item 38]
Item 38. A vector production system comprising a recombinant cell engineered to encode at least a first recombinase under the control of an inducible promoter, the cell comprising the expression vector according to any one of items 1 to 37.
[Item 39]
Item 39. The vector production system of Item 38, wherein the inducible promoter is thermally regulated, chemically regulated, IPTG regulated, glucose regulated, arabinose inducible, T7 polymerase regulated, cold shock inducible, pH inducible, or a combination thereof.
[Item 40]
the first recombinase is selected from telN and tel; and
40. The vector production system of claim 38 or 39, wherein the expression vector incorporates the target sequence for at least the first recombinase.
[Item 41]
The vector production system of any one of claims 38 to 40, wherein the recombinant cell is further designed to encode a nuclease genome editing system, and the expression vector further comprises a backbone sequence comprising a cleavage site for the nuclease genome editing system.
[Item 42]
Item 42. The vector production system of item 41, wherein the nuclease genome editing system is a CRISPR nuclease system comprising a Cas nuclease and a gRNA, and the expression vector comprises a target sequence for the gRNA within the backbone sequence.
[Item 43]
43. A method for producing a bacterial sequence-free vector, comprising the step of incubating the vector production system according to any one of claims 38 to 42 under conditions suitable for expression of the first recombinase.
[Item 44]
43. A method for producing a vector free of bacterial sequences, comprising incubating the vector production system of claim 41 or 42 under conditions suitable for expression of the first recombinase and the nuclease genome editing system.
[Item 45]
45. The method of paragraph 43 or 44, further comprising harvesting the bacterial sequence-free vector.
[Item 46]
46. A bacterial sequence-free vector produced by the method according to any one of paragraphs 43 to 45.
[Item 47]
A vector free of bacterial sequences comprising an expression cassette comprising a nucleic acid sequence encoding a recombinant protein comprising a conserved amino acid sequence derived from a virus fused to an immunogenic amino acid sequence, wherein the protein expressed intracellularly from the expression cassette is capable of forming a VLP.
[Item 48]
48. The bacterial sequence-free vector of claim 47, wherein the immunogenic amino acid sequence is from the same virus as the conserved amino acid sequence.
[Item 49]
49. The bacterial sequence-free vector of paragraph 47 or 48, wherein the conserved amino acid sequence is derived from a viral glycoprotein.
[Item 50]
50. The bacterial sequence-free vector of claim 49, wherein the immunogenic amino acid sequence is derived from the same viral glycoprotein.
[Item 51]
51. The bacterial sequence-free vector of any one of paragraphs 47 to 50, wherein the expression cassette further comprises a nucleic acid sequence encoding a viral envelope protein and/or a nucleic acid sequence encoding a viral matrix protein.
[Item 52]
52. The bacterial sequence-free vector of claim 51, wherein the viral envelope protein and/or the viral matrix protein is from the same virus as the conserved amino acid sequence.
[Item 53]
53. The bacterial sequence-free vector of any one of paragraphs 47 to 52, wherein the conserved amino acid sequence, the immunogenic amino acid sequence, the viral envelope protein, and/or the viral matrix protein are consensus sequences.
[Item 54]
54. The bacterial sequence free vector of any one of paragraphs 47 to 53, wherein the recombinant protein is capable of stimulating an immune response against the virus that includes neutralizing antibodies.
[Item 55]
55. The bacterial sequence free vector of any one of paragraphs 47 to 54, wherein the recombinant protein is capable of stimulating a Th1 cell mediated immune response against the virus.
[Item 56]
56. The bacterial sequence-free vector of paragraph 54 or 55, wherein the immune response is cross-reactive to related viruses or related strains.
[Item 57]
57. The bacterial sequence-free vector of any one of paragraphs 47 to 56, wherein the recombinant protein excludes amino acid sequences derived from the virus that stimulate an immune response involving non-neutralizing antibodies and/or stimulates a Th2 cell-mediated immune response.
[Item 58]
58. The bacterial sequence-free vector of any one of paragraphs 47 to 57, wherein the expression cassette comprises a single open reading frame comprising a nucleic acid sequence encoding a self-cleaving peptide between each nucleic acid sequence encoding a protein.
[Item 59]
59. The bacterial sequence free vector of any one of paragraphs 47 to 58, wherein the virus is a coronavirus, influenza virus, human immunodeficiency virus, human papilloma virus, hepatitis virus, or an oncolytic virus.
[Item 60]
60. The bacterial sequence-free vector of claim 59, wherein the virus is a coronavirus.
[Item 61]
61. The bacterial sequence-free vector of claim 60, wherein the coronavirus is COVID-19.
[Item 62]
62. The bacterial sequence-free vector of paragraph 60 or 61, wherein the expression cassette comprises a nucleic acid sequence encoding a recombinant protein comprising conserved and immunogenic amino acid sequences derived from a coronavirus M protein, a coronavirus E protein, and a coronavirus S protein.
[Item 63]
63. The bacterial sequence free vector of paragraph 62, wherein the conserved amino acid sequences are derived from the S protein S2' cleavage site and IFP.
[Item 64]
64. The bacterial sequence-free vector of paragraph 62 or 63, wherein the conserved amino acid sequence comprises SEQ ID NO:12.
[Item 65]
65. The bacterial sequence-free vector of any one of paragraphs 62 to 64, wherein the immunogenic amino acid sequence is derived from the S protein RBD.
[Item 66]
66. The bacterial sequence-free vector of any one of paragraphs 62-65, wherein the immunogenic amino acid sequence is at least about 90% identical to SEQ ID NO:11.
[Item 67]
67. The bacterial sequence-free vector of any one of paragraphs 62 to 66, wherein the recombinant protein further comprises a TM domain sequence from the S protein.
[Item 68]
68. The bacterial sequence free vector of any one of paragraphs 62-67, wherein the recombinant protein excludes amino acid sequences from the S protein that stimulate an immune response involving non-neutralizing antibodies and/or stimulates a Th2 cell mediated immune response.
[Item 69]
69. The bacterial sequence-free vector of any one of paragraphs 62-68, wherein the amino acid sequence of the recombinant protein is at least about 90% identical to SEQ ID NO:55.
[Item 70]
70. The bacterial sequence-free vector of any one of paragraphs 62-69, wherein the expression cassette comprises a single open reading frame translated as an amino acid sequence that is at least about 90% identical to SEQ ID NO:57.
[Item 71]
71. The bacterial sequence-free vector of any one of paragraphs 62 to 70, wherein the recombinant protein is capable of stimulating an immune response against COVID-19.
[Item 72]
72. The bacterial sequence-free vector of any one of paragraphs 62-71, wherein the recombinant protein is capable of stimulating a Th1 cell-mediated immune response against COVID-19.
[Item 73]
73. The bacterial sequence-free vector of clause 71 or 72, wherein the immune response is cross-reactive to other coronaviruses.
[Item 74]
74. The bacterial sequence-free vector of claim 73, wherein the immune response is cross-reactive to other severe acute respiratory syndrome coronaviruses and/or human betacoronaviruses.
[Item 75]
75. The bacterial sequence-free vector of any one of paragraphs 47 to 74, further comprising at least one enhancer sequence flanking said expression cassette.
[Item 76]
76. The bacterial sequence-free vector of Paragraph 75, wherein the at least one enhancer sequence is at least two enhancer sequences.
[Item 77]
77. The bacterial sequence-free vector of paragraph 75 or 76, wherein at least one enhancer sequence is an SV40 enhancer sequence.
[Item 78]
78. The bacterial sequence-free vector of any one of paragraphs 47 to 77, comprising circular covalently closed ends.
[Item 79]
78. The bacterial sequence-free vector of any one of paragraphs 47 to 77, comprising linear, covalently linked closed ends.
[Item 80]
A polynucleotide encoding an amino acid sequence that is at least about 90% identical to SEQ ID NO:57.
[Item 81]
A recombinant cell comprising the expression vector of any one of paragraphs 1 to 37 or the bacterial sequence-free vector of any one of paragraphs 46 to 79.
[Item 82]
82. A method of producing a VLP comprising culturing the recombinant cell of Paragraph 81 under conditions suitable for production of said VLP from said expression vector or said vector free of bacterial sequences.
[Item 83]
83. The method of claim 82, further comprising the step of isolating said VLP.
[Item 84]
84. The method of claim 83, wherein the isolating step is carried out by affinity purification.
[Item 85]
85. The method of any one of paragraphs 82 to 84, wherein the VLP is produced by the expression vector of any one of paragraphs 14 to 37 or the bacterial sequence free vector of any one of paragraphs 60 to 79.
[Item 86]
86. The method of claim 85, wherein the affinity purification involves angiotensin converting enzyme 2 (ACE2) receptor peptide or an anti-S protein monoclonal antibody.
[Item 87]
87. The method of claim 86, wherein the ACE2 receptor peptide comprises an amino acid sequence that is at least about 90 identical to the amino acid sequence of SEQ ID NO:70.
[Item 88]
88. The method of claim 86 or 87, wherein the ACE2 receptor peptide comprises a biotin acceptor peptide (BAP) tag at the C-terminus or N-terminus of the peptide.
[Item 89]
89. The method of claim 88, wherein the BAP tag comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:71.
[Item 90]
90. The method of any one of paragraphs 86 to 89, wherein the ACE2 receptor peptide or anti-S protein monoclonal antibody is biotinylated and immobilized on streptavidin-coated beads.
[Item 91]
91. The method of any one of claims 84 to 90, wherein the affinity purification comprises microfluidics and/or chromatography.
[Item 92]
92. A VLP produced by the method according to any one of items 82 to 91.
[Item 93]
A VLP comprising a recombinant protein containing a conserved amino acid sequence from a virus fused to an immunogenic amino acid sequence.
[Item 94]
94. The VLP of claim 93, wherein the immunogenic amino acid sequence is derived from the same virus as the conserved amino acid sequence.
[Item 95]
95. The VLP of claim 93 or 94, wherein the conserved amino acid sequence is derived from a viral glycoprotein.
[Item 96]
96. The VLP of any one of paragraphs 93 to 95, wherein the immunogenic amino acid sequences are derived from the same viral glycoprotein.
[Item 97]
97. The VLP of any one of paragraphs 93 to 96, further comprising a viral envelope protein and/or a viral matrix protein.
[Item 98]
98. The VLP of claim 97, wherein the viral envelope protein and/or the viral matrix protein are derived from the same virus as the conserved amino acid sequence.
[Item 99]
99. The VLP of any one of paragraphs 93 to 98, wherein the conserved amino acid sequence, the immunogenic amino acid sequence, the viral envelope protein, and/or the viral matrix protein are consensus sequences.
[Item 100]
100. The VLP of any one of paragraphs 93-99, wherein the recombinant protein is capable of stimulating an immune response against the virus, including neutralizing antibodies.
[Item 101]
101. The VLP of any one of paragraphs 93-100, wherein the recombinant protein is capable of stimulating a Th1 cell-mediated immune response against the virus.
[Item 102]
102. The VLP of paragraph 100 or 101, wherein the immune response is cross-reactive to related viruses or related strains.
[Item 103]
103. The VLP of any one of paragraphs 93-102, wherein the recombinant protein excludes amino acid sequences derived from the virus that stimulate an immune response involving non-neutralizing antibodies and/or stimulates a Th2 cell-mediated immune response.
[Item 104]
104. The VLP of any one of paragraphs 93 to 103, wherein the virus is a coronavirus, influenza virus, human immunodeficiency virus, human papilloma virus, hepatitis virus, or an oncolytic virus.
[Item 105]
105. The VLP of paragraph 104, wherein the virus is a coronavirus.
[Item 106]
106. The VLP of paragraph 105, wherein the coronavirus is COVID-19.
[Item 107]
107. The VLP of claim 105 or 106, comprising recombinant proteins comprising conserved and immunogenic amino acid sequences derived from a coronavirus membrane (M) protein, a coronavirus envelope (E) protein, and a coronavirus spike (S) protein.
[Item 108]
108. The VLP of Paragraph 107, wherein the conserved amino acid sequences are derived from the S protein S2' cleavage site and internal fusion peptide (IFP).
[Item 109]
109. The VLP of paragraph 107 or 108, wherein the conserved amino acid sequence comprises SEQ ID NO:12.
[Item 110]
110. The VLP of any one of paragraphs 107 to 109, wherein the immunogenic amino acid sequence is derived from the S protein receptor binding domain (RBD).
[Item 111]
111. The VLP of any one of paragraphs 107 to 110, wherein the immunogenic amino acid sequence is at least about 90% identical to SEQ ID NO:11.
[Item 112]
112. The VLP of any one of paragraphs 107-111, wherein the recombinant protein further comprises a transmembrane (TM) domain sequence from the S protein.
[Item 113]
113. The VLP of any one of paragraphs 107-112, wherein the recombinant protein excludes amino acid sequences from the S protein that stimulate immune responses involving non-neutralizing antibodies and/or stimulate Th2 cell-mediated immune responses.
[Item 114]
114. The VLP of any one of paragraphs 107-113, wherein the amino acid sequence of the recombinant protein is at least about 90% identical to SEQ ID NO:55.
[Item 115]
A VLP comprising a recombinant protein at least about 90% identical to SEQ ID NO:55, an M protein at least about 90% identical to SEQ ID NO:1, and an E protein at least about 90% identical to SEQ ID NO:3.
[Item 116]
116. The VLP of any one of paragraphs 107 to 115, wherein the recombinant protein is capable of stimulating an immune response against COVID-19.
[Item 117]
117. The VLP of any one of paragraphs 107 to 116, wherein the recombinant protein is capable of stimulating a Th1 cell-mediated immune response against COVID-19.
[Item 118]
118. The VLP of paragraph 116 or 117, wherein the immune response is cross-reactive to other coronaviruses.
[Item 119]
The VLP of paragraph 118, wherein the immune response is cross-reactive to other severe acute respiratory syndrome coronaviruses and/or human betacoronaviruses.
[Item 120]
120. A composition comprising the expression vector of any one of paragraphs 1-37, the bacterial sequence-free vector of any one of paragraphs 46-79, or the virus-like particle of any one of paragraphs 92-119.
[Item 121]
121. The composition of claim 120, further comprising a delivery agent.
[Item 122]
122. The composition of claim 121, wherein the delivery agent is a nanoparticle.
[Item 123]
123. The composition of claim 121 or 122, wherein the delivery agent comprises a targeting ligand.
[Item 124]
124. The composition of claim 123, wherein the targeting ligand comprises an S protein peptide.
[Item 125]
The composition of Paragraph 124, wherein the S protein peptide comprises an amino acid sequence at least about 90% identical to any one of SEQ ID NOs:76-99.
[Item 126]
127. A method of treating a viral infection in a subject comprising the expression vector of any one of paragraphs 1-37, the bacterial sequence-free vector of any one of paragraphs 46-79, the VLP of any one of paragraphs 92-119, or the composition of any one of paragraphs 120-125, wherein intracellular expression of the expression vector or the bacterial sequence-free vector produces a VLP.
[Item 127]
127. The method of claim 126, wherein the administering step is performed by parenteral or non-parenteral administration.
[Item 128]
128. The method of claim 127, wherein said administering step is carried out by oral, pulmonary, intranasal, intravenous, epidermal, transdermal, subcutaneous, intramuscular, or intraperitoneal injection, or by inhalation.
[Item 129]
129. The method of any one of paragraphs 126 to 128, wherein the VLP stimulates an immune response in the subject that includes neutralizing antibodies against the viral infection.
[Item 130]
130. The method of any one of paragraphs 126 to 129, wherein the VLP stimulates a Th1 cell-mediated immune response in the subject against the viral infection.
[Item 131]
131. The method of claim 129 or 130, wherein the immune response is cross-reactive to related viruses or related strains.
[Item 132]
132. The method of any one of paragraphs 126-131, wherein said VLP does not stimulate an immune response in said subject that includes non-neutralizing antibodies and/or does not stimulate a Th2 cell-mediated immune response in said subject.
[Item 133]
133. The method of any one of paragraphs 126 to 132, wherein the VLP cross-competes with the infectious virus for binding to a viral receptor.
[Item 134]
134. The method of paragraph 133, wherein the VLP cross-competes with related viruses or strains for binding to the viral receptor.
[Item 135]
135. The method of any one of paragraphs 126 to 134, wherein the viral infection is a coronavirus, influenza virus, human immunodeficiency virus, human papilloma virus, hepatitis virus, or an oncolytic virus.
[Item 136]
136. The method of claim 135, wherein the viral infection is a coronavirus.
[Item 137]
137. The method of claim 136, wherein the viral infection is COVID-19.
[Item 138]
138. The method of claim 137, wherein the VLP stimulates an immune response in the subject, including neutralizing antibodies against COVID-19.
[Item 139]
139. The method of paragraph 137 or 138, wherein the VLP stimulates a Th1 cell-mediated immune response in a subject against COVID-19.
[Item 140]
140. The method of claim 138 or 139, wherein the immune response is cross-reactive to other coronaviruses.
[Item 141]
141. The method of claim 140, wherein the immune response is cross-reactive to other severe acute respiratory syndrome coronaviruses and/or human betacoronaviruses.
[Item 142]
142. The method of any one of paragraphs 137-141, wherein said VLP does not stimulate an immune response in said subject that includes non-neutralizing antibodies and/or does not stimulate a Th2 cell-mediated immune response in said subject.
[Item 143]
143. The method of any one of paragraphs 137 to 142, wherein the administering step is performed by inhalation.
[Item 144]
144. The method of any one of paragraphs 137 to 143, wherein the VLP cross-competes with COVID-19 for binding to the ACE2 receptor, neuropilin-1, or other receptor.
[Item 145]
145. The method of paragraph 144, wherein the VLP cross-competes with other coronaviruses for binding to the ACE2 receptor, neuropilin-1, and/or other receptors.
[Item 146]
146. The method of paragraph 145, wherein the VLP cross-competes with other severe acute respiratory syndrome coronaviruses and/or human betacoronaviruses for binding to the ACE2 receptor, neuropilin-1, and/or other receptors.

Claims (24)

an expression cassette comprising a nucleic acid sequence encoding a recombinant protein comprising a conserved amino acid sequence derived from a virus fused to an immunogenic amino acid sequence;
target sequences for a first recombinase flanking said expression cassette;
and one or more additional target sequences for one or more additional recombinases integrated within the non-binding regions of the target sequence for the first recombinase,
An expression vector, wherein a protein expressed from said expression cassette in a cell is capable of forming a virus-like particle (VLP). A bacterial sequence-free vector comprising an expression cassette containing a nucleic acid sequence encoding a recombinant protein comprising a conserved amino acid sequence derived from a virus fused to an immunogenic amino acid sequence, wherein the protein expressed intracellularly from the expression cassette is capable of forming a VLP. (a) the immunogenic amino acid sequence is derived from the same virus as the conserved amino acid sequence;
(b) the conserved amino acid sequence is derived from a viral glycoprotein, and optionally the immunogenic amino acid sequence is derived from the same viral glycoprotein;
(c) the expression cassette further comprises a nucleic acid sequence encoding a viral envelope protein and/or a nucleic acid sequence encoding a viral matrix protein, optionally wherein the viral envelope protein and/or the viral matrix protein are derived from the same virus as the conserved amino acid sequence;
(d) the conserved amino acid sequence, the immunogenic amino acid sequence, the viral envelope protein, and/or the viral matrix protein are consensus sequences;
(e) the recombinant protein is capable of stimulating an immune response against the virus, including neutralizing antibodies, and optionally, the immune response is cross-reactive against related viruses or related strains;
(f) the recombinant protein is capable of stimulating a Th1 cell-mediated immune response against the virus, optionally which immune response is cross-reactive against related viruses or related strains;
(g) the recombinant protein excludes viral-derived amino acid sequences that stimulate immune responses including non-neutralizing antibodies and/or stimulate Th2 cell-mediated immune responses;
(h) the expression cassette comprises a single open reading frame comprising a nucleic acid sequence encoding a self-cleaving peptide between each nucleic acid sequence encoding a protein;
(i) the virus is a coronavirus, influenza virus, human immunodeficiency virus, human papilloma virus, hepatitis virus, or an oncolytic virus; or (j) any combination of the above;
Optionally, the virus is a coronavirus, and further optionally, the coronavirus is COVID-19.
3. An expression vector according to claim 1 or a bacterial sequence-free vector according to claim 2. The expression vector or bacterial sequence-free vector of claim 3, wherein the expression cassette comprises a nucleic acid sequence encoding a recombinant protein comprising conserved and immunogenic amino acid sequences from a coronavirus membrane (M) protein, a coronavirus envelope (E) protein, and a coronavirus spike (S) protein. (a) the conserved amino acid sequence is derived from the S protein S2' cleavage site and the internal fusion peptide (IFP);
(b) the conserved amino acid sequence comprises SEQ ID NO: 12;
(c) the immunogenic amino acid sequence is derived from the S protein receptor binding domain (RBD);
(d) the immunogenic amino acid sequence is (i) at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:11, or (ii) comprises SEQ ID NO:11;
(e) the recombinant protein further comprises a transmembrane (TM) domain sequence from the S protein;
(f) the recombinant protein excludes amino acid sequences from the S protein that stimulate immune responses including non-neutralizing antibodies and/or stimulate Th2 cell-mediated immune responses;
(g) the amino acid sequence of the recombinant protein is (i) at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:55, or (ii) comprises SEQ ID NO:55;
(h) the nucleic acid sequence of the recombinant protein is (i) at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:56, or (ii) comprises SEQ ID NO:56;
(i) the expression cassette is (i) at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO:57; or (ii) comprises a single open reading frame translated as an amino acid sequence that includes SEQ ID NO:57.
(j) the expression cassette is (i) at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:58, or (ii) comprises a single open reading frame that includes SEQ ID NO:58;
(k) the recombinant protein is capable of stimulating an immune response against COVID-19, optionally, the immune response is cross-reactive to other coronaviruses, and further optionally, the immune response is cross-reactive to other severe acute respiratory syndrome coronaviruses and/or human betacoronaviruses;
(l) the recombinant protein is capable of stimulating a Th1 cell-mediated immune response against COVID-19, optionally wherein said immune response is cross-reactive to other coronaviruses, and further optionally wherein said immune response is cross-reactive to other severe acute respiratory syndrome coronaviruses and/or human betacoronaviruses; or
(m) any combination of the above;
5. The expression vector of claim 4 or a vector free of bacterial sequences. The expression vector of any one of claims 1 to 5, wherein the target sequence for the first recombinase and the one or more additional target sequences for the one or more additional recombinases are selected from the group consisting of PY54 pal site, N15 telRL site, loxP site, φK02 telRL site, FRT site, phiC31 attP site, and λ attP site, and optionally the expression vector comprises each of the target sequences, and further optionally the expression vector comprises a Tel recombinase pal site and the telRL, loxP, and FRT recombinase target binding sequences integrated within the pal site. A bacterial sequence-free vector according to any one of claims 2 to 5, having circular covalently linked closed ends. A bacterial sequence-free vector according to any one of claims 2 to 5, having linear, covalently linked closed ends. A vector production system comprising a recombinant cell designed to encode at least a first recombinase under the control of an inducible promoter, the cell comprising an expression vector according to any one of claims 1 or 3 to 6, optionally wherein the inducible promoter is thermally regulated, chemically regulated, IPTG regulated, glucose regulated, arabinose induced, T7 polymerase regulated, cold shock induced, pH induced, or a combination thereof, and further optionally wherein the first recombinase is selected from telN and tel, and the expression vector incorporates a target sequence for at least the first recombinase. A method for producing a vector free of bacterial sequences, comprising incubating the vector production system of claim 9 under conditions suitable for expression of the first recombinase, and optionally further comprising harvesting the vector free of bacterial sequences. (i) A polynucleotide that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO:55 or 57, or (ii) encodes an amino acid sequence that includes SEQ ID NO:55 or 57. (i) A polynucleotide comprising a nucleic acid sequence that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO:56 or 58, or (ii) comprises a nucleic acid sequence that includes SEQ ID NO:56 or 58. A recombinant cell comprising an expression vector according to any one of claims 1 or 3 to 6, or a vector not containing a bacterial sequence according to any one of claims 2 to 5, 7 or 8. 14. A method for producing a VLP comprising culturing the recombinant cell of claim 13 under conditions suitable for the production of VLPs from an expression vector or a vector free of bacterial sequences, optionally further comprising the step of isolating the VLPs, optionally said isolating step being performed by affinity purification, optionally said affinity purification comprising an angiotensin converting enzyme 2 (ACE2) receptor peptide or an anti-S protein monoclonal antibody, optionally
(a) an ACE2 receptor peptide that is (i) at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO:70, or (ii) comprises an amino acid sequence that includes SEQ ID NO:70;
(b) the ACE2 receptor peptide comprises a biotin acceptor peptide (BAP) tag at the C-terminus or N-terminus of the peptide, optionally wherein the BAP tag is (i) at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to the amino acid sequence of SEQ ID NO:71, or (ii) comprises an amino acid sequence comprising SEQ ID NO:71;
(c) the ACE2 receptor peptide or the anti-S protein monoclonal antibody is biotinylated and immobilized on streptavidin-coated beads, or (d) any combination of the above, said method for producing the VLP. A VLP comprising a recombinant protein containing a conserved amino acid sequence derived from a virus fused to an immunogenic amino acid sequence. (a) the immunogenic amino acid sequence is derived from the same virus as the conserved amino acid sequence;
(b) the conserved amino acid sequence is derived from a viral glycoprotein, and optionally the immunogenic amino acid sequence is derived from the same viral glycoprotein;
(c) the VLP further comprises a viral envelope protein and/or a viral matrix protein, optionally wherein the viral envelope protein and/or the viral matrix protein are derived from the same virus as the conserved amino acid sequence;
(d) the conserved amino acid sequence, the immunogenic amino acid sequence, the viral envelope protein, and/or the viral matrix protein are consensus sequences;
(e) the recombinant protein is capable of stimulating an immune response against said virus, including neutralizing antibodies, and optionally, said immune response is cross-reactive against related viruses or related strains;
(f) the recombinant protein is capable of stimulating a Th1 cell-mediated immune response against said virus, and optionally, the immune response is cross-reactive against related viruses or related strains;
(g) the recombinant protein excludes amino acid sequences derived from the virus that stimulate immune responses including non-neutralizing antibodies and/or stimulate Th2 cell-mediated immune responses;
(h) the virus is a coronavirus, influenza virus, human immunodeficiency virus, human papilloma virus, hepatitis virus, or an oncolytic virus; or
(i) any combination of the above,
Optionally, the virus is a coronavirus, and further optionally, the coronavirus is COVID-19. 17. The VLP of claim 16, comprising recombinant proteins comprising conserved and immunogenic amino acid sequences from a coronavirus membrane (M) protein, a coronavirus envelope (E) protein, and a coronavirus spike (S) protein, optionally comprising:
(a) the conserved amino acid sequence is derived from the S protein S2' cleavage site and the internal fusion peptide (IFP);
(b) the conserved amino acid sequence comprises SEQ ID NO: 12;
(c) the immunogenic amino acid sequence is derived from the S protein receptor binding domain (RBD);
(d) the immunogenic amino acid sequence is (i) at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:11, or (ii) comprises SEQ ID NO:11;
(e) the recombinant protein further comprises a transmembrane (TM) domain sequence derived from the S protein;
(f) the recombinant protein excludes amino acid sequences from the S protein that stimulate immune responses including non-neutralizing antibodies and/or stimulate Th2 cell-mediated immune responses;
(g) the amino acid sequence of the recombinant protein is (i) at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:55, or (ii) comprises SEQ ID NO:55;
(h) the amino acid sequence of the recombinant protein is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:55, the amino acid sequence of the M protein is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:1, and the amino acid sequence of the E protein is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO:3;
(i) the amino acid sequence of the recombinant protein comprises SEQ ID NO:55, the amino acid sequence of the M protein comprises SEQ ID NO:1, and the amino acid sequence of the E protein comprises SEQ ID NO:3;
(j) the recombinant protein is capable of stimulating an immune response against COVID-19, optionally, said immune response being cross-reactive to other coronaviruses, and further optionally, said immune response being cross-reactive to other severe acute respiratory syndrome coronaviruses and/or human betacoronaviruses;
(k) the recombinant protein is capable of stimulating a Th1 cell-mediated immune response against COVID-19, optionally wherein said immune response is cross-reactive to other coronaviruses, and further optionally wherein said immune response is cross-reactive to other severe acute respiratory syndrome coronaviruses and/or human betacoronaviruses; or
(l) any combination of the above;
The VLP. (i) A polypeptide that is at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to SEQ ID NO:55 or 57, or (ii) comprises an amino acid sequence that includes SEQ ID NO:55 or 57. A composition comprising an expression vector according to any one of claims 1 or 3 to 6, a vector free of bacterial sequences according to any one of claims 2 to 5, 7 or 8, a VLP according to any one of claims 15 to 17, or a polypeptide according to claim 18, optionally further comprising a delivery agent, optionally the delivery agent being a nanoparticle, optionally the delivery agent comprising a targeting ligand, optionally the targeting ligand comprising an S protein peptide, optionally the S protein peptide being (i) at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical to any one of SEQ ID NOs: 76 to 99, or (ii) comprising an amino acid sequence comprising any one of SEQ ID NOs: 76 to 99. The expression vector of any one of claims 1 or 3-6, the bacterial sequence-free vector of any one of claims 2-5, 7 or 8, the VLP of any one of claims 15-17, the polypeptide of claim 18, or the composition of claim 19 for use in VLP treatment of a viral infection in a subject, optionally wherein the viral infection is a coronavirus, influenza virus, human immunodeficiency virus, human papilloma virus, hepatitis virus, or oncolytic virus, and further optionally wherein the viral infection is COVID-19. 21. The expression vector, bacterial sequence-free vector, VLP, polypeptide or composition of claim 20, formulated for parenteral or non-parenteral administration, optionally formulated for oral, pulmonary, intranasal, intravenous, epidermal, transdermal, subcutaneous, intramuscular or intraperitoneal administration or for inhalation. 22. The expression vector, bacterial sequence free vector, VLP, polypeptide or composition of claim 20 or 21, wherein the VLP stimulates an immune response in the subject comprising neutralizing antibodies against the viral infection and/or the VLP stimulates a Th1 cell mediated immune response in the subject against the viral infection, optionally the immune response being cross-reactive against related viruses or related strains. (a) the VLP does not stimulate an immune response in said subject that includes non-neutralizing antibodies and/or does not stimulate a Th2 cell-mediated immune response in said subject; and/or
(b) the VLP cross-competes with the infectious virus for binding to a viral receptor, optionally, the VLP cross-competes with a related virus or strain for binding to the viral receptor;
23. An expression vector, bacterial sequence free vector, VLP, polypeptide or composition according to any one of claims 20 to 22. (a) the VLPs stimulate an immune response in a subject including neutralizing antibodies against COVID-19, optionally, said immune response is cross-reactive to other coronaviruses, and further optionally, said immune response is cross-reactive to other severe acute respiratory syndrome coronaviruses and/or human betacoronaviruses;
(b) the VLPs stimulate a Th1 cell-mediated immune response in a subject against COVID-19, optionally, said immune response being cross-reactive to other coronaviruses, and further optionally, said immune response being cross-reactive to other severe acute respiratory syndrome coronaviruses and/or human betacoronaviruses.
(c) the VLP does not stimulate an immune response in said subject that includes non-neutralizing antibodies and/or does not stimulate a Th2 cell-mediated immune response in said subject;
(d) formulated for inhalation; and/or
(e) the VLP cross-competes with COVID-19 for binding to the ACE2 receptor, neuropilin-1, or other receptors, optionally, the VLP cross-competes with other coronaviruses for binding to the ACE2 receptor, neuropilin-1, and/or other receptors, and further optionally, the VLP cross-competes with other severe acute respiratory syndrome coronaviruses and/or human betacoronaviruses for binding to the ACE2 receptor, neuropilin-1, and/or other receptors;
24. An expression vector, bacterial sequence free vector, VLP, polypeptide or composition according to any one of claims 20 to 23.