WO2023230481A1 - Orthopoxvirus vaccines - Google Patents
Orthopoxvirus vaccines Download PDFInfo
- Publication number
- WO2023230481A1 WO2023230481A1 PCT/US2023/067368 US2023067368W WO2023230481A1 WO 2023230481 A1 WO2023230481 A1 WO 2023230481A1 US 2023067368 W US2023067368 W US 2023067368W WO 2023230481 A1 WO2023230481 A1 WO 2023230481A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- orthopoxvirus
- composition
- protein
- mrna
- subject
- Prior art date
Links
- 241000700629 Orthopoxvirus Species 0.000 title claims abstract description 76
- 229960005486 vaccine Drugs 0.000 title abstract description 123
- 238000000034 method Methods 0.000 claims abstract description 77
- 208000005871 monkeypox Diseases 0.000 claims abstract description 18
- 239000000427 antigen Substances 0.000 claims description 199
- 102000036639 antigens Human genes 0.000 claims description 196
- 108091007433 antigens Proteins 0.000 claims description 196
- 239000000203 mixture Substances 0.000 claims description 171
- 150000002632 lipids Chemical class 0.000 claims description 88
- 239000002105 nanoparticle Substances 0.000 claims description 69
- 108700010766 orthopoxvirus proteins Proteins 0.000 claims description 69
- -1 amino lipid Chemical class 0.000 claims description 67
- 102000040430 polynucleotide Human genes 0.000 claims description 63
- 108091033319 polynucleotide Proteins 0.000 claims description 63
- 239000002157 polynucleotide Substances 0.000 claims description 62
- 108700026244 Open Reading Frames Proteins 0.000 claims description 59
- 241000700605 Viruses Species 0.000 claims description 49
- 229920002477 rna polymer Polymers 0.000 claims description 32
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 26
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 18
- 239000012634 fragment Substances 0.000 claims description 17
- 206010069586 Orthopox virus infection Diseases 0.000 claims description 14
- 229930182558 Sterol Natural products 0.000 claims description 14
- 150000003432 sterols Chemical class 0.000 claims description 14
- 235000003702 sterols Nutrition 0.000 claims description 14
- 235000012000 cholesterol Nutrition 0.000 claims description 13
- 230000002163 immunogen Effects 0.000 claims description 13
- 238000007385 chemical modification Methods 0.000 claims description 12
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 claims description 10
- UVBYMVOUBXYSFV-XUTVFYLZSA-N 1-methylpseudouridine Chemical compound O=C1NC(=O)N(C)C=C1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 UVBYMVOUBXYSFV-XUTVFYLZSA-N 0.000 claims description 10
- UVBYMVOUBXYSFV-UHFFFAOYSA-N 1-methylpseudouridine Natural products O=C1NC(=O)N(C)C=C1C1C(O)C(O)C(CO)O1 UVBYMVOUBXYSFV-UHFFFAOYSA-N 0.000 claims description 7
- 229940125904 compound 1 Drugs 0.000 claims description 7
- JFBCSFJKETUREV-LJAQVGFWSA-N 1,2-ditetradecanoyl-sn-glycerol Chemical compound CCCCCCCCCCCCCC(=O)OC[C@H](CO)OC(=O)CCCCCCCCCCCCC JFBCSFJKETUREV-LJAQVGFWSA-N 0.000 claims description 2
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 2
- 108700021021 mRNA Vaccine Proteins 0.000 abstract description 33
- 241000700647 Variola virus Species 0.000 abstract description 21
- 229940126582 mRNA vaccine Drugs 0.000 abstract description 16
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 124
- 150000007523 nucleic acids Chemical class 0.000 description 113
- 102000039446 nucleic acids Human genes 0.000 description 111
- 108020004707 nucleic acids Proteins 0.000 description 111
- 102000053602 DNA Human genes 0.000 description 104
- 108020004414 DNA Proteins 0.000 description 103
- 125000003729 nucleotide group Chemical group 0.000 description 93
- 108090000623 proteins and genes Proteins 0.000 description 89
- 102000004169 proteins and genes Human genes 0.000 description 77
- 235000018102 proteins Nutrition 0.000 description 76
- 239000002773 nucleotide Substances 0.000 description 69
- 108090000765 processed proteins & peptides Proteins 0.000 description 55
- 230000028993 immune response Effects 0.000 description 51
- 230000003053 immunization Effects 0.000 description 42
- 210000004027 cell Anatomy 0.000 description 41
- 102000004196 processed proteins & peptides Human genes 0.000 description 38
- 108020003589 5' Untranslated Regions Proteins 0.000 description 32
- 229920001184 polypeptide Polymers 0.000 description 32
- 108020004999 messenger RNA Proteins 0.000 description 30
- 125000006273 (C1-C3) alkyl group Chemical group 0.000 description 27
- 108020004705 Codon Proteins 0.000 description 26
- 125000000217 alkyl group Chemical group 0.000 description 26
- 230000003472 neutralizing effect Effects 0.000 description 26
- 125000003342 alkenyl group Chemical group 0.000 description 25
- 125000006592 (C2-C3) alkenyl group Chemical group 0.000 description 24
- 108020005345 3' Untranslated Regions Proteins 0.000 description 24
- 239000002777 nucleoside Substances 0.000 description 24
- 150000001875 compounds Chemical class 0.000 description 23
- 108091023045 Untranslated Region Proteins 0.000 description 21
- 208000015181 infectious disease Diseases 0.000 description 20
- 125000005647 linker group Chemical group 0.000 description 20
- 125000003835 nucleoside group Chemical group 0.000 description 20
- 238000006243 chemical reaction Methods 0.000 description 19
- 241000700627 Monkeypox virus Species 0.000 description 18
- 230000000890 antigenic effect Effects 0.000 description 18
- 108010033040 Histones Proteins 0.000 description 17
- 229940022005 RNA vaccine Drugs 0.000 description 17
- 125000000623 heterocyclic group Chemical group 0.000 description 17
- 108091026890 Coding region Proteins 0.000 description 16
- 108010076504 Protein Sorting Signals Proteins 0.000 description 15
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 15
- 229910052739 hydrogen Inorganic materials 0.000 description 15
- 238000004519 manufacturing process Methods 0.000 description 15
- 230000014616 translation Effects 0.000 description 15
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 14
- 230000006870 function Effects 0.000 description 14
- 230000003612 virological effect Effects 0.000 description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 13
- 235000001014 amino acid Nutrition 0.000 description 13
- 150000001413 amino acids Chemical class 0.000 description 13
- 230000014509 gene expression Effects 0.000 description 13
- 125000001072 heteroaryl group Chemical group 0.000 description 13
- 238000000338 in vitro Methods 0.000 description 13
- 229920001223 polyethylene glycol Polymers 0.000 description 12
- 108020005176 AU Rich Elements Proteins 0.000 description 11
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 11
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 11
- 241000700618 Vaccinia virus Species 0.000 description 11
- 230000001580 bacterial effect Effects 0.000 description 11
- 238000013518 transcription Methods 0.000 description 11
- 230000027455 binding Effects 0.000 description 10
- 238000001727 in vivo Methods 0.000 description 10
- 230000004048 modification Effects 0.000 description 10
- 238000012986 modification Methods 0.000 description 10
- 238000010606 normalization Methods 0.000 description 10
- 230000000069 prophylactic effect Effects 0.000 description 10
- 150000003839 salts Chemical class 0.000 description 10
- 238000003786 synthesis reaction Methods 0.000 description 10
- 230000035897 transcription Effects 0.000 description 10
- 238000013519 translation Methods 0.000 description 10
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 9
- 241000282412 Homo Species 0.000 description 9
- 108091036407 Polyadenylation Proteins 0.000 description 9
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 9
- 230000015556 catabolic process Effects 0.000 description 9
- 238000006731 degradation reaction Methods 0.000 description 9
- 230000036039 immunity Effects 0.000 description 9
- 238000011282 treatment Methods 0.000 description 9
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 description 8
- 102000008857 Ferritin Human genes 0.000 description 8
- 108050000784 Ferritin Proteins 0.000 description 8
- 238000008416 Ferritin Methods 0.000 description 8
- 108090000288 Glycoproteins Proteins 0.000 description 8
- 102000003886 Glycoproteins Human genes 0.000 description 8
- 108010052285 Membrane Proteins Proteins 0.000 description 8
- 108091081024 Start codon Proteins 0.000 description 8
- 102220536912 Transcription factor JunD_A35R_mutation Human genes 0.000 description 8
- 101100000230 Vaccinia virus (strain Copenhagen) A35R gene Proteins 0.000 description 8
- 101100000231 Vaccinia virus (strain Western Reserve) VACWR158 gene Proteins 0.000 description 8
- 125000003118 aryl group Chemical group 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 8
- 238000009472 formulation Methods 0.000 description 8
- 230000001939 inductive effect Effects 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 239000011541 reaction mixture Substances 0.000 description 8
- 238000006467 substitution reaction Methods 0.000 description 8
- 230000001225 therapeutic effect Effects 0.000 description 8
- 238000002255 vaccination Methods 0.000 description 8
- 102000018697 Membrane Proteins Human genes 0.000 description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 description 7
- 101710137500 T7 RNA polymerase Proteins 0.000 description 7
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 7
- 238000004422 calculation algorithm Methods 0.000 description 7
- 229910052799 carbon Inorganic materials 0.000 description 7
- 108020001507 fusion proteins Proteins 0.000 description 7
- 102000037865 fusion proteins Human genes 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 239000008194 pharmaceutical composition Substances 0.000 description 7
- 239000000546 pharmaceutical excipient Substances 0.000 description 7
- 238000012545 processing Methods 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 7
- 229940045145 uridine Drugs 0.000 description 7
- 125000004400 (C1-C12) alkyl group Chemical group 0.000 description 6
- 108010068996 6,7-dimethyl-8-ribityllumazine synthase Proteins 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 6
- 239000002202 Polyethylene glycol Substances 0.000 description 6
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 6
- 239000002671 adjuvant Substances 0.000 description 6
- 125000004429 atom Chemical group 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 229940104302 cytosine Drugs 0.000 description 6
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000010172 mouse model Methods 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000000746 purification Methods 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 101150080480 A27L gene Proteins 0.000 description 5
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical class NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 5
- 101100439665 Arabidopsis thaliana SWI2 gene Proteins 0.000 description 5
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 5
- 108091034057 RNA (poly(A)) Proteins 0.000 description 5
- 101100268532 Vaccinia virus (strain Western Reserve) VACWR150 gene Proteins 0.000 description 5
- 108010067390 Viral Proteins Proteins 0.000 description 5
- 239000004480 active ingredient Substances 0.000 description 5
- 239000013543 active substance Substances 0.000 description 5
- 229960005305 adenosine Drugs 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 210000004899 c-terminal region Anatomy 0.000 description 5
- 238000004113 cell culture Methods 0.000 description 5
- 238000012217 deletion Methods 0.000 description 5
- 230000037430 deletion Effects 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 125000000592 heterocycloalkyl group Chemical group 0.000 description 5
- 230000005847 immunogenicity Effects 0.000 description 5
- 230000003834 intracellular effect Effects 0.000 description 5
- 230000000670 limiting effect Effects 0.000 description 5
- 230000007935 neutral effect Effects 0.000 description 5
- 238000005457 optimization Methods 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000000087 stabilizing effect Effects 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- ZAYHVCMSTBRABG-UHFFFAOYSA-N 5-Methylcytidine Natural products O=C1N=C(N)C(C)=CN1C1C(O)C(O)C(CO)O1 ZAYHVCMSTBRABG-UHFFFAOYSA-N 0.000 description 4
- ZAYHVCMSTBRABG-JXOAFFINSA-N 5-methylcytidine Chemical compound O=C1N=C(N)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 ZAYHVCMSTBRABG-JXOAFFINSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 125000000882 C2-C6 alkenyl group Chemical group 0.000 description 4
- 102000055765 ELAV-Like Protein 1 Human genes 0.000 description 4
- 102100022823 Histone RNA hairpin-binding protein Human genes 0.000 description 4
- 101000825762 Homo sapiens Histone RNA hairpin-binding protein Proteins 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 241001183012 Modified Vaccinia Ankara virus Species 0.000 description 4
- 229930185560 Pseudouridine Natural products 0.000 description 4
- PTJWIQPHWPFNBW-UHFFFAOYSA-N Pseudouridine C Natural products OC1C(O)C(CO)OC1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-UHFFFAOYSA-N 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 238000005571 anion exchange chromatography Methods 0.000 description 4
- WGDUUQDYDIIBKT-UHFFFAOYSA-N beta-Pseudouridine Natural products OC1OC(CN2C=CC(=O)NC2=O)C(O)C1O WGDUUQDYDIIBKT-UHFFFAOYSA-N 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 239000003937 drug carrier Substances 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 4
- 210000001808 exosome Anatomy 0.000 description 4
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 4
- 125000005842 heteroatom Chemical group 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- 150000003833 nucleoside derivatives Chemical class 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 230000008488 polyadenylation Effects 0.000 description 4
- 238000004007 reversed phase HPLC Methods 0.000 description 4
- 239000007790 solid phase Substances 0.000 description 4
- 235000000346 sugar Nutrition 0.000 description 4
- 229910052717 sulfur Inorganic materials 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 239000001226 triphosphate Substances 0.000 description 4
- 235000011178 triphosphate Nutrition 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 229940035893 uracil Drugs 0.000 description 4
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 description 3
- 101800001779 2'-O-methyltransferase Proteins 0.000 description 3
- 229930024421 Adenine Natural products 0.000 description 3
- 101710132601 Capsid protein Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 3
- 102000002067 Protein Subunits Human genes 0.000 description 3
- 108010001267 Protein Subunits Proteins 0.000 description 3
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 3
- 230000024932 T cell mediated immunity Effects 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 229960000643 adenine Drugs 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000005875 antibody response Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 230000007969 cellular immunity Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 210000002443 helper t lymphocyte Anatomy 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 229940031551 inactivated vaccine Drugs 0.000 description 3
- 230000002458 infectious effect Effects 0.000 description 3
- 231100000518 lethal Toxicity 0.000 description 3
- 230000001665 lethal effect Effects 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 229940023143 protein vaccine Drugs 0.000 description 3
- PTJWIQPHWPFNBW-GBNDHIKLSA-N pseudouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-GBNDHIKLSA-N 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 229940031626 subunit vaccine Drugs 0.000 description 3
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 3
- 230000032258 transport Effects 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- 229960004854 viral vaccine Drugs 0.000 description 3
- FVXDQWZBHIXIEJ-LNDKUQBDSA-N 1,2-di-[(9Z,12Z)-octadecadienoyl]-sn-glycero-3-phosphocholine Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC FVXDQWZBHIXIEJ-LNDKUQBDSA-N 0.000 description 2
- SNKAWJBJQDLSFF-NVKMUCNASA-N 1,2-dioleoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC SNKAWJBJQDLSFF-NVKMUCNASA-N 0.000 description 2
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 2
- KVUXYQHEESDGIJ-UHFFFAOYSA-N 10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthrene-3,16-diol Chemical compound C1CC2CC(O)CCC2(C)C2C1C1CC(O)CC1(C)CC2 KVUXYQHEESDGIJ-UHFFFAOYSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 description 2
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 101100180402 Caenorhabditis elegans jun-1 gene Proteins 0.000 description 2
- 241001137864 Camelpox virus Species 0.000 description 2
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 108030002463 Dye decolorizing peroxidases Proteins 0.000 description 2
- 241000206602 Eukaryota Species 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerol Natural products OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 2
- XKMLYUALXHKNFT-UUOKFMHZSA-N Guanosine-5'-triphosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XKMLYUALXHKNFT-UUOKFMHZSA-N 0.000 description 2
- 102100039869 Histone H2B type F-S Human genes 0.000 description 2
- 101001035372 Homo sapiens Histone H2B type F-S Proteins 0.000 description 2
- 101001045218 Homo sapiens Peroxisomal multifunctional enzyme type 2 Proteins 0.000 description 2
- 241000928771 Horsepox virus Species 0.000 description 2
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 241000700639 Parapoxvirus Species 0.000 description 2
- 108091093037 Peptide nucleic acid Proteins 0.000 description 2
- 241000700625 Poxviridae Species 0.000 description 2
- 241001455645 Rabbitpox virus Species 0.000 description 2
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 2
- 230000005867 T cell response Effects 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 101150114197 TOP gene Proteins 0.000 description 2
- 241000204666 Thermotoga maritima Species 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 206010046865 Vaccinia virus infection Diseases 0.000 description 2
- 241001137865 Volepox virus Species 0.000 description 2
- FHHZHGZBHYYWTG-INFSMZHSSA-N [(2r,3s,4r,5r)-5-(2-amino-7-methyl-6-oxo-3h-purin-9-ium-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl [[[(2r,3s,4r,5r)-5-(2-amino-6-oxo-3h-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl] phosphate Chemical compound N1C(N)=NC(=O)C2=C1[N+]([C@H]1[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=C(C(N=C(N)N4)=O)N=C3)O)O1)O)=CN2C FHHZHGZBHYYWTG-INFSMZHSSA-N 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000002238 attenuated effect Effects 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- LGJMUZUPVCAVPU-UHFFFAOYSA-N beta-Sitostanol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CC)C(C)C)C1(C)CC2 LGJMUZUPVCAVPU-UHFFFAOYSA-N 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 125000001369 canonical nucleoside group Chemical group 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 238000001818 capillary gel electrophoresis Methods 0.000 description 2
- 125000002091 cationic group Chemical group 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical group O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 238000007306 functionalization reaction Methods 0.000 description 2
- 229960004956 glycerylphosphorylcholine Drugs 0.000 description 2
- 238000011194 good manufacturing practice Methods 0.000 description 2
- 229940029575 guanosine Drugs 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 208000002672 hepatitis B Diseases 0.000 description 2
- 230000004727 humoral immunity Effects 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 238000000126 in silico method Methods 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 229940124590 live attenuated vaccine Drugs 0.000 description 2
- 229940023012 live-attenuated vaccine Drugs 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000004807 localization Effects 0.000 description 2
- UYEUUXMDVNYCAM-UHFFFAOYSA-N lumazine Chemical compound N1=CC=NC2=NC(O)=NC(O)=C21 UYEUUXMDVNYCAM-UHFFFAOYSA-N 0.000 description 2
- 235000018977 lysine Nutrition 0.000 description 2
- 125000003588 lysine group Chemical class [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 238000007069 methylation reaction Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 229940031348 multivalent vaccine Drugs 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 210000004909 pre-ejaculatory fluid Anatomy 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000002028 premature Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 108020001580 protein domains Proteins 0.000 description 2
- 238000000275 quality assurance Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- NLQLSVXGSXCXFE-UHFFFAOYSA-N sitosterol Natural products CC=C(/CCC(C)C1CC2C3=CCC4C(C)C(O)CCC4(C)C3CCC2(C)C1)C(C)C NLQLSVXGSXCXFE-UHFFFAOYSA-N 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 229940113082 thymine Drugs 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- 230000014621 translational initiation Effects 0.000 description 2
- 229940125575 vaccine candidate Drugs 0.000 description 2
- 208000007089 vaccinia Diseases 0.000 description 2
- 108010027510 vaccinia virus capping enzyme Proteins 0.000 description 2
- 230000008673 vomiting Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 2
- KZJWDPNRJALLNS-VPUBHVLGSA-N (-)-beta-Sitosterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]([C@H](CC[C@@H](C(C)C)CC)C)CC4)CC3)CC=2)CC1 KZJWDPNRJALLNS-VPUBHVLGSA-N 0.000 description 1
- JTERLNYVBOZRHI-PPBJBQABSA-N (2-aminoethoxy)[(2r)-2,3-bis[(5z,8z,11z,14z)-icosa-5,8,11,14-tetraenoyloxy]propoxy]phosphinic acid Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC JTERLNYVBOZRHI-PPBJBQABSA-N 0.000 description 1
- XLKQWAMTMYIQMG-SVUPRYTISA-N (2-{[(2r)-2,3-bis[(4z,7z,10z,13z,16z,19z)-docosa-4,7,10,13,16,19-hexaenoyloxy]propyl phosphonato]oxy}ethyl)trimethylazanium Chemical compound CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CC\C=C/C\C=C/C\C=C/C\C=C/C\C=C/C\C=C/CC XLKQWAMTMYIQMG-SVUPRYTISA-N 0.000 description 1
- CSVWWLUMXNHWSU-UHFFFAOYSA-N (22E)-(24xi)-24-ethyl-5alpha-cholest-22-en-3beta-ol Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(CC)C(C)C)C1(C)CC2 CSVWWLUMXNHWSU-UHFFFAOYSA-N 0.000 description 1
- RQOCXCFLRBRBCS-UHFFFAOYSA-N (22E)-cholesta-5,7,22-trien-3beta-ol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CCC(C)C)CCC33)C)C3=CC=C21 RQOCXCFLRBRBCS-UHFFFAOYSA-N 0.000 description 1
- WCGUUGGRBIKTOS-GPOJBZKASA-N (3beta)-3-hydroxyurs-12-en-28-oic acid Chemical compound C1C[C@H](O)C(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@@]5(C(O)=O)CC[C@@H](C)[C@H](C)[C@H]5C4=CC[C@@H]3[C@]21C WCGUUGGRBIKTOS-GPOJBZKASA-N 0.000 description 1
- LVNGJLRDBYCPGB-LDLOPFEMSA-N (R)-1,2-distearoylphosphatidylethanolamine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[NH3+])OC(=O)CCCCCCCCCCCCCCCCC LVNGJLRDBYCPGB-LDLOPFEMSA-N 0.000 description 1
- SSCDRSKJTAQNNB-DWEQTYCFSA-N 1,2-di-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphoethanolamine Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC SSCDRSKJTAQNNB-DWEQTYCFSA-N 0.000 description 1
- LZLVZIFMYXDKCN-QJWFYWCHSA-N 1,2-di-O-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC LZLVZIFMYXDKCN-QJWFYWCHSA-N 0.000 description 1
- KILNVBDSWZSGLL-KXQOOQHDSA-N 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCC KILNVBDSWZSGLL-KXQOOQHDSA-N 0.000 description 1
- DSNRWDQKZIEDDB-SQYFZQSCSA-N 1,2-dioleoyl-sn-glycero-3-phospho-(1'-sn-glycerol) Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@@H](O)CO)OC(=O)CCCCCCC\C=C/CCCCCCCC DSNRWDQKZIEDDB-SQYFZQSCSA-N 0.000 description 1
- MWRBNPKJOOWZPW-NYVOMTAGSA-N 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine zwitterion Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/CCCCCCCC MWRBNPKJOOWZPW-NYVOMTAGSA-N 0.000 description 1
- PDXQSLIBLQMPJS-FDDDBJFASA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-(methoxymethyl)pyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(COC)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 PDXQSLIBLQMPJS-FDDDBJFASA-N 0.000 description 1
- WTJKGGKOPKCXLL-VYOBOKEXSA-N 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC WTJKGGKOPKCXLL-VYOBOKEXSA-N 0.000 description 1
- OZNBTMLHSVZFLR-GWTDSMLYSA-N 2-amino-9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-3h-purin-6-one;6-amino-1h-pyrimidin-2-one Chemical compound NC=1C=CNC(=O)N=1.C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OZNBTMLHSVZFLR-GWTDSMLYSA-N 0.000 description 1
- XLPHMKQBBCKEFO-DHYROEPTSA-N 2-azaniumylethyl [(2r)-2,3-bis(3,7,11,15-tetramethylhexadecanoyloxy)propyl] phosphate Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)CC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CC(C)CCCC(C)CCCC(C)CCCC(C)C XLPHMKQBBCKEFO-DHYROEPTSA-N 0.000 description 1
- SLQKYSPHBZMASJ-QKPORZECSA-N 24-methylene-cholest-8-en-3β-ol Chemical compound C([C@@]12C)C[C@H](O)C[C@@H]1CCC1=C2CC[C@]2(C)[C@@H]([C@H](C)CCC(=C)C(C)C)CC[C@H]21 SLQKYSPHBZMASJ-QKPORZECSA-N 0.000 description 1
- KLEXDBGYSOIREE-UHFFFAOYSA-N 24xi-n-propylcholesterol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)CCC(CCC)C(C)C)C1(C)CC2 KLEXDBGYSOIREE-UHFFFAOYSA-N 0.000 description 1
- 101710176159 32 kDa protein Proteins 0.000 description 1
- IZFJAICCKKWWNM-JXOAFFINSA-N 4-amino-1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-methoxypyrimidin-2-one Chemical compound O=C1N=C(N)C(OC)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IZFJAICCKKWWNM-JXOAFFINSA-N 0.000 description 1
- AMMRPAYSYYGRKP-BGZDPUMWSA-N 5-[(2s,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1-ethylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)N(CC)C=C1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 AMMRPAYSYYGRKP-BGZDPUMWSA-N 0.000 description 1
- ZXIATBNUWJBBGT-JXOAFFINSA-N 5-methoxyuridine Chemical compound O=C1NC(=O)C(OC)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 ZXIATBNUWJBBGT-JXOAFFINSA-N 0.000 description 1
- 101150020560 A32L gene Proteins 0.000 description 1
- 102100027573 ATP synthase subunit alpha, mitochondrial Human genes 0.000 description 1
- 241000254032 Acrididae Species 0.000 description 1
- 241000180579 Arca Species 0.000 description 1
- 241000203069 Archaea Species 0.000 description 1
- 241000238421 Arthropoda Species 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- OILXMJHPFNGGTO-NRHJOKMGSA-N Brassicasterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@](C)([C@H]([C@@H](/C=C/[C@H](C(C)C)C)C)CC4)CC3)CC=2)CC1 OILXMJHPFNGGTO-NRHJOKMGSA-N 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 210000004366 CD4-positive T-lymphocyte Anatomy 0.000 description 1
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 1
- SGNBVLSWZMBQTH-FGAXOLDCSA-N Campesterol Natural products O[C@@H]1CC=2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]([C@H](CC[C@H](C(C)C)C)C)CC4)CC3)CC=2)CC1 SGNBVLSWZMBQTH-FGAXOLDCSA-N 0.000 description 1
- 101710167800 Capsid assembly scaffolding protein Proteins 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 241000700628 Chordopoxvirinae Species 0.000 description 1
- LPZCCMIISIBREI-MTFRKTCUSA-N Citrostadienol Natural products CC=C(CC[C@@H](C)[C@H]1CC[C@H]2C3=CC[C@H]4[C@H](C)[C@@H](O)CC[C@]4(C)[C@H]3CC[C@]12C)C(C)C LPZCCMIISIBREI-MTFRKTCUSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000700626 Cowpox virus Species 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 101710082494 DNA protection during starvation protein Proteins 0.000 description 1
- 238000011238 DNA vaccination Methods 0.000 description 1
- ARVGMISWLZPBCH-UHFFFAOYSA-N Dehydro-beta-sitosterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)CCC(CC)C(C)C)CCC33)C)C3=CC=C21 ARVGMISWLZPBCH-UHFFFAOYSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- GZDFHIJNHHMENY-UHFFFAOYSA-N Dimethyl dicarbonate Chemical compound COC(=O)OC(=O)OC GZDFHIJNHHMENY-UHFFFAOYSA-N 0.000 description 1
- 102000016662 ELAV Proteins Human genes 0.000 description 1
- 108010053101 ELAV Proteins Proteins 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- DNVPQKQSNYMLRS-NXVQYWJNSA-N Ergosterol Natural products CC(C)[C@@H](C)C=C[C@H](C)[C@H]1CC[C@H]2C3=CC=C4C[C@@H](O)CC[C@]4(C)[C@@H]3CC[C@]12C DNVPQKQSNYMLRS-NXVQYWJNSA-N 0.000 description 1
- 241000701533 Escherichia virus T4 Species 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 108091092566 Extrachromosomal DNA Proteins 0.000 description 1
- 101710189104 Fibritin Proteins 0.000 description 1
- 102000003983 Flavoproteins Human genes 0.000 description 1
- 108010057573 Flavoproteins Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 102000048120 Galactokinases Human genes 0.000 description 1
- 108700023157 Galactokinases Proteins 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 241000699694 Gerbillinae Species 0.000 description 1
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 1
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- BTEISVKTSQLKST-UHFFFAOYSA-N Haliclonasterol Natural products CC(C=CC(C)C(C)(C)C)C1CCC2C3=CC=C4CC(O)CCC4(C)C3CCC12C BTEISVKTSQLKST-UHFFFAOYSA-N 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 241000590002 Helicobacter pylori Species 0.000 description 1
- 102100027685 Hemoglobin subunit alpha Human genes 0.000 description 1
- 108091005902 Hemoglobin subunit alpha Proteins 0.000 description 1
- 102100021519 Hemoglobin subunit beta Human genes 0.000 description 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- 101000936262 Homo sapiens ATP synthase subunit alpha, mitochondrial Proteins 0.000 description 1
- 101001050288 Homo sapiens Transcription factor Jun Proteins 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 102000001974 Hyaluronidases Human genes 0.000 description 1
- 102000018251 Hypoxanthine Phosphoribosyltransferase Human genes 0.000 description 1
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 101710203526 Integrase Proteins 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 108091027974 Mature messenger RNA Proteins 0.000 description 1
- 102000029749 Microtubule Human genes 0.000 description 1
- 108091022875 Microtubule Proteins 0.000 description 1
- 241000700559 Molluscipoxvirus Species 0.000 description 1
- 102000004364 Myogenin Human genes 0.000 description 1
- 108010056785 Myogenin Proteins 0.000 description 1
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
- WIHSZOXPODIZSW-KJIWEYRQSA-N PE(18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15Z)) Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)CCCCCCC\C=C/C\C=C/C\C=C/CC WIHSZOXPODIZSW-KJIWEYRQSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000002508 Peptide Elongation Factors Human genes 0.000 description 1
- 108010068204 Peptide Elongation Factors Proteins 0.000 description 1
- 208000005228 Pericardial Effusion Diseases 0.000 description 1
- 102100022587 Peroxisomal multifunctional enzyme type 2 Human genes 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 101710130420 Probable capsid assembly scaffolding protein Proteins 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 108020005073 RNA Cap Analogs Proteins 0.000 description 1
- 101710086015 RNA ligase Proteins 0.000 description 1
- 108010065868 RNA polymerase SP6 Proteins 0.000 description 1
- 230000004570 RNA-binding Effects 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000700638 Raccoonpox virus Species 0.000 description 1
- 206010037888 Rash pustular Diseases 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 102000002278 Ribosomal Proteins Human genes 0.000 description 1
- 108010000605 Ribosomal Proteins Proteins 0.000 description 1
- 230000018199 S phase Effects 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 206010039509 Scab Diseases 0.000 description 1
- 101710204410 Scaffold protein Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 241000321597 Skunkpox virus Species 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 241000723792 Tobacco etch virus Species 0.000 description 1
- XYNPYHXGMWJBLV-VXPJTDKGSA-N Tomatidine Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)CC[C@H](O)C[C@@H]4CC[C@H]3[C@@H]2C1)C)[C@@H]1C)[C@@]11CC[C@H](C)CN1 XYNPYHXGMWJBLV-VXPJTDKGSA-N 0.000 description 1
- 101001023030 Toxoplasma gondii Myosin-D Proteins 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 102100023132 Transcription factor Jun Human genes 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102100040247 Tumor necrosis factor Human genes 0.000 description 1
- 108010007780 U7 Small Nuclear Ribonucleoprotein Proteins 0.000 description 1
- 108091026823 U7 small nuclear RNA Proteins 0.000 description 1
- OILXMJHPFNGGTO-ZRUUVFCLSA-N UNPD197407 Natural products C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)C=C[C@H](C)C(C)C)[C@@]1(C)CC2 OILXMJHPFNGGTO-ZRUUVFCLSA-N 0.000 description 1
- HZYXFRGVBOPPNZ-UHFFFAOYSA-N UNPD88870 Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)=CCC(CC)C(C)C)C1(C)CC2 HZYXFRGVBOPPNZ-UHFFFAOYSA-N 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 108010065667 Viral Matrix Proteins Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 108010042365 Virus-Like Particle Vaccines Proteins 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- 241000700574 Yatapoxvirus Species 0.000 description 1
- NJFCSWSRXWCWHV-USYZEHPZSA-N [(2R)-2,3-bis(octadec-1-enoxy)propyl] 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCCC=COC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC=CCCCCCCCCCCCCCCCC NJFCSWSRXWCWHV-USYZEHPZSA-N 0.000 description 1
- SUTHKQVOHCMCCF-QZNUWAOFSA-N [(2r)-3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-docosa-2,4,6,8,10,12-hexaenoyloxypropyl] docosa-2,4,6,8,10,12-hexaenoate Chemical compound CCCCCCCCCC=CC=CC=CC=CC=CC=CC(=O)OC[C@H](COP(O)(=O)OCCN)OC(=O)C=CC=CC=CC=CC=CC=CCCCCCCCCC SUTHKQVOHCMCCF-QZNUWAOFSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000000370 acceptor Substances 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 150000003838 adenosines Chemical class 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 229940087168 alpha tocopherol Drugs 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 210000001742 aqueous humor Anatomy 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 238000000429 assembly Methods 0.000 description 1
- 230000000712 assembly Effects 0.000 description 1
- 230000006472 autoimmune response Effects 0.000 description 1
- 108010028263 bacteriophage T3 RNA polymerase Proteins 0.000 description 1
- SLQKYSPHBZMASJ-UHFFFAOYSA-N bastadin-1 Natural products CC12CCC(O)CC1CCC1=C2CCC2(C)C(C(C)CCC(=C)C(C)C)CCC21 SLQKYSPHBZMASJ-UHFFFAOYSA-N 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- MJVXAPPOFPTTCA-UHFFFAOYSA-N beta-Sistosterol Natural products CCC(CCC(C)C1CCC2C3CC=C4C(C)C(O)CCC4(C)C3CCC12C)C(C)C MJVXAPPOFPTTCA-UHFFFAOYSA-N 0.000 description 1
- NJKOMDUNNDKEAI-UHFFFAOYSA-N beta-sitosterol Natural products CCC(CCC(C)C1CCC2(C)C3CC=C4CC(O)CCC4C3CCC12C)C(C)C NJKOMDUNNDKEAI-UHFFFAOYSA-N 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 230000006287 biotinylation Effects 0.000 description 1
- 238000007413 biotinylation Methods 0.000 description 1
- 210000004952 blastocoel Anatomy 0.000 description 1
- 208000027499 body ache Diseases 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- OILXMJHPFNGGTO-ZAUYPBDWSA-N brassicasterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)/C=C/[C@H](C)C(C)C)[C@@]1(C)CC2 OILXMJHPFNGGTO-ZAUYPBDWSA-N 0.000 description 1
- 235000004420 brassicasterol Nutrition 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- SGNBVLSWZMBQTH-PODYLUTMSA-N campesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](C)C(C)C)[C@@]1(C)CC2 SGNBVLSWZMBQTH-PODYLUTMSA-N 0.000 description 1
- 235000000431 campesterol Nutrition 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 150000001783 ceramides Chemical class 0.000 description 1
- 210000002939 cerumen Anatomy 0.000 description 1
- 210000003756 cervix mucus Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 210000001268 chyle Anatomy 0.000 description 1
- 210000004913 chyme Anatomy 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000011258 core-shell material Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000002726 cyst fluid Anatomy 0.000 description 1
- 108010009442 cytochrome b245 Proteins 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000004163 cytometry Methods 0.000 description 1
- 210000005220 cytoplasmic tail Anatomy 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000012350 deep sequencing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 239000005549 deoxyribonucleoside Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000000368 destabilizing effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 150000001982 diacylglycerols Chemical class 0.000 description 1
- 125000005265 dialkylamine group Chemical group 0.000 description 1
- 150000001985 dialkylglycerols Chemical class 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 238000001085 differential centrifugation Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000002612 dispersion medium Substances 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 229940126534 drug product Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- DNVPQKQSNYMLRS-SOWFXMKYSA-N ergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H](CC[C@]3([C@H]([C@H](C)/C=C/[C@@H](C)C(C)C)CC[C@H]33)C)C3=CC=C21 DNVPQKQSNYMLRS-SOWFXMKYSA-N 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 150000002211 flavins Chemical class 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000002873 global sequence alignment Methods 0.000 description 1
- 108060003196 globin Proteins 0.000 description 1
- 102000018146 globin Human genes 0.000 description 1
- 230000036433 growing body Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 229940037467 helicobacter pylori Drugs 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 230000028996 humoral immune response Effects 0.000 description 1
- 229960002773 hyaluronidase Drugs 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 239000012642 immune effector Substances 0.000 description 1
- 238000012151 immunohistochemical method Methods 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- DTOSIQBPPRVQHS-PDBXOOCHSA-M linolenate Chemical compound CC\C=C/C\C=C/C\C=C/CCCCCCCC([O-])=O DTOSIQBPPRVQHS-PDBXOOCHSA-M 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- VLBPIWYTPAXCFJ-XMMPIXPASA-N lysophosphatidylcholine O-16:0/0:0 Chemical compound CCCCCCCCCCCCCCCCOC[C@@H](O)COP([O-])(=O)OCC[N+](C)(C)C VLBPIWYTPAXCFJ-XMMPIXPASA-N 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000013178 mathematical model Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 210000004914 menses Anatomy 0.000 description 1
- 210000004688 microtubule Anatomy 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 210000001819 pancreatic juice Anatomy 0.000 description 1
- 150000002972 pentoses Chemical class 0.000 description 1
- 210000004912 pericardial fluid Anatomy 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 239000008024 pharmaceutical diluent Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 150000008103 phosphatidic acids Chemical class 0.000 description 1
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 210000004910 pleural fluid Anatomy 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 229940021993 prophylactic vaccine Drugs 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 210000004908 prostatic fluid Anatomy 0.000 description 1
- 235000019419 proteases Nutrition 0.000 description 1
- 230000020978 protein processing Effects 0.000 description 1
- 125000000561 purinyl group Chemical group N1=C(N=C2N=CNC2=C1)* 0.000 description 1
- 210000004915 pus Anatomy 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 229940126583 recombinant protein vaccine Drugs 0.000 description 1
- 230000003014 reinforcing effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000011506 response to oxidative stress Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000012340 reverse transcriptase PCR Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 229960002477 riboflavin Drugs 0.000 description 1
- 235000019192 riboflavin Nutrition 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 210000003935 rough endoplasmic reticulum Anatomy 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 210000002374 sebum Anatomy 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 229910052814 silicon oxide Inorganic materials 0.000 description 1
- PWRIIDWSQYQFQD-UHFFFAOYSA-N sisunine Natural products CC1CCC2(NC1)OC3CC4C5CCC6CC(CCC6(C)C5CCC4(C)C3C2C)OC7OC(CO)C(OC8OC(CO)C(O)C(OC9OC(CO)C(O)C(O)C9OC%10OC(CO)C(O)C(O)C%10O)C8O)C(O)C7O PWRIIDWSQYQFQD-UHFFFAOYSA-N 0.000 description 1
- KZJWDPNRJALLNS-VJSFXXLFSA-N sitosterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CC[C@@H](CC)C(C)C)[C@@]1(C)CC2 KZJWDPNRJALLNS-VJSFXXLFSA-N 0.000 description 1
- 235000015500 sitosterol Nutrition 0.000 description 1
- 229950005143 sitosterol Drugs 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 206010040882 skin lesion Diseases 0.000 description 1
- 231100000444 skin lesion Toxicity 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- IUVFCFQZFCOKRC-IPKKNMRRSA-M sodium;[(2r)-2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl] 2,3-dihydroxypropyl phosphate Chemical compound [Na+].CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C/CCCCCCCC IUVFCFQZFCOKRC-IPKKNMRRSA-M 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 239000012798 spherical particle Substances 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 229940032091 stigmasterol Drugs 0.000 description 1
- HCXVJBMSMIARIN-PHZDYDNGSA-N stigmasterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)/C=C/[C@@H](CC)C(C)C)[C@@]1(C)CC2 HCXVJBMSMIARIN-PHZDYDNGSA-N 0.000 description 1
- 235000016831 stigmasterol Nutrition 0.000 description 1
- BFDNMXAIBMJLBB-UHFFFAOYSA-N stigmasterol Natural products CCC(C=CC(C)C1CCCC2C3CC=C4CC(O)CCC4(C)C3CCC12C)C(C)C BFDNMXAIBMJLBB-UHFFFAOYSA-N 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 210000001138 tear Anatomy 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- 230000008719 thickening Effects 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 229960000984 tocofersolan Drugs 0.000 description 1
- XYNPYHXGMWJBLV-OFMODGJOSA-N tomatidine Natural products O[C@@H]1C[C@H]2[C@@](C)([C@@H]3[C@H]([C@H]4[C@@](C)([C@H]5[C@@H](C)[C@]6(O[C@H]5C4)NC[C@@H](C)CC6)CC3)CC2)CC1 XYNPYHXGMWJBLV-OFMODGJOSA-N 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 108010087967 type I signal peptidase Proteins 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- PLSAJKYPRJGMHO-UHFFFAOYSA-N ursolic acid Natural products CC1CCC2(CCC3(C)C(C=CC4C5(C)CCC(O)C(C)(C)C5CCC34C)C2C1C)C(=O)O PLSAJKYPRJGMHO-UHFFFAOYSA-N 0.000 description 1
- 229940096998 ursolic acid Drugs 0.000 description 1
- 230000007501 viral attachment Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 210000004916 vomit Anatomy 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
- 239000002076 α-tocopherol Substances 0.000 description 1
- 235000004835 α-tocopherol Nutrition 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/24011—Poxviridae
- C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
- C12N2710/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- Poxvirus (a member of the Poxviridae) is a double-stranded DNA virus that infects both humans and animals. Poxviruses are divided into two subfamilies based on host range: the Acrididae (Chordopoxviridae) family includes four genera, orthopoxvirus genus (Orthopoxvirus), Parapoxvirus (Parapoxvirus), molluscum pox virus (Molluscipoxvirus) and Yatapoxvirus, that are known to infect humans.
- the Orthopoxvirus genera includes a number of genetically related and morphologically identical viruses, including variola virus (VARV), camel pox virus (CMLV), vaccinia virus (CPXV), apoplexy virus (ECTV), horse pox virus (HPXV), and monkeypox virus (MPXV), rabbit pox virus (RPXV), raccoon pox virus, skunk pox virus, gerbil pox virus, Uasin Gishu disease virus, vaccinia virus (VACV), smallpox virus (VARV), and volepox virus (VPV). At least four viruses are known to infect humans: VARV, VACV, MPXV, and CPXV.
- compositions e.g., vaccines
- mRNA messenger ribonucleic acid
- the mRNA molecules described herein are used to express key antigenic components of the virus (e.g., protein ectodomains of A35L and B6R derived from extracellular virus (EV), M1R and A29L derived from mature virus (MV)) that are efficient at inducing protective immunity when used individually or in combination as an immunogenic composition or vaccine to protect people from infection by the natural virus and/or to reduce symptoms if infected.
- virus e.g., protein ectodomains of A35L and B6R derived from extracellular virus (EV), M1R and A29L derived from mature virus (MV)
- compositions comprising an mRNA encoding a functional domain of an orthopoxvirus (e.g., smallpox, monkeypox) capable of inducing an immune response, such as a neutralizing antibody response, to an orthopoxvirus (e.g., smallpox, monkeypox).
- the mRNA is formulated in a lipid nanoparticle.
- Some aspects of the disclosure provide a composition, comprising a first messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame (ORF) encoding a first orthopoxvirus protein and a lipid nanoparticle.
- mRNA messenger ribonucleic acid
- ORF open reading frame
- the composition further comprises a second mRNA polynucleotide comprising an ORF encoding a second orthopoxvirus protein. In some embodiments, the composition further comprises a third mRNA polynucleotide comprising an ORF encoding a third orthopoxvirus protein. In some embodiments, the composition further comprises a fourth mRNA polynucleotide comprising an ORF encoding a fourth orthopoxvirus protein. In some embodiments, the first orthopoxvirus protein comprises a mature virus (MV) orthopoxvirus protein or an extracellular enveloped virus (EV) orthopoxvirus protein.
- MV mature virus
- EV extracellular enveloped virus
- the second orthopoxvirus protein comprises a MV orthopoxvirus protein or an EV orthopoxvirus protein.
- the third orthopoxvirus protein comprises an MV orthopoxvirus protein or an EV orthopoxvirus protein.
- the fourth orthopoxvirus protein comprises an MV orthopoxvirus protein or an EV orthopoxvirus protein.
- the MV orthopoxvirus protein is A29L or M1R.
- the EV orthopoxvirus protein is B6R or A35R.
- the first orthopoxvirus protein comprises A27L
- the second orthopoxvirus protein comprises M1R
- the third orthopoxvirus protein comprises B6R
- the fourth orthopoxvirus protein comprises A35R.
- the disclosure in some aspects, provides a composition comprising a first messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame (ORF) encoding a first orthopoxvirus protein, and a lipid nanoparticle, wherein the first orthopoxvirus protein is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 7-34 or an immunogenic fragment thereof.
- mRNA messenger ribonucleic acid
- ORF open reading frame
- the composition further comprises a second mRNA polynucleotide comprising an ORF encoding a second orthopoxvirus protein, wherein the second orthopoxvirus protein is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 7-34 or an immunogenic fragment thereof.
- the composition further comprises a third mRNA polynucleotide comprising an ORF encoding a third orthopoxvirus protein, wherein the third orthopoxvirus protein is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 7-34 or an immunogenic fragment thereof.
- the composition further comprises a fourth mRNA polynucleotide comprising an ORF encoding a fourth orthopoxvirus protein, wherein the fourth orthopoxvirus protein is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 7-34 or an immunogenic fragment thereof.
- the orthopoxvirus protein is at least 90%, at least 95%, at least 97%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 7-34.
- the orthopoxvirus protein is identical to any one of the amino acid sequences of SEQ ID NOs: 7-34.
- the ORF comprises a sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical to any one of SEQ ID NOs: 35-62. In some embodiments, the ORF comprises a sequence that identical to any one of SEQ ID NOs: 35-62.
- the first, second, third, and/or fourth mRNA comprises a chemical modification. In some embodiments, the first, second, third, and/or fourth mRNA is fully modified. In some embodiments, the chemical modification is 1-methylpseudouridine.
- the lipid nanoparticle comprises a molar ratio of 20-60% ionizable amino lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises 1-5 mol% PEG-modified lipid; 10-20 mol% non- cationic lipid; 35-45 mol% sterol; and 40-50 mol% ionizable cationic lipid.
- the PEG-modified lipid is 1,2 dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG2000 DMG), the non-cationic lipid is 1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC), the sterol is cholesterol; and the ionizable cationic lipid has the structure of Compound 1: (Compound 1).
- the disclosure provides a method for vaccinating a subject, comprising administering to the subject any one of the compositions described herein. In some embodiments, the method prevents an orthopoxvirus infection in the subject. In some embodiments, the method reduces the severity of an orthopoxvirus infection in the subject.
- the subject is seronegative for an orthopoxvirus. In some embodiments, the subject is seropositive for an orthopoxvirus.
- the disclosure provides multivalent RNA composition, comprising at least two messenger ribonucleic acid (mRNA) polynucleotides, each comprising an open reading frame (ORF) encoding a monkeypox antigen and a lipid nanoparticle.
- mRNA messenger ribonucleic acid
- ORF open reading frame
- the monkeypox antigen comprises a mature virus (MV) orthopoxvirus protein and/or an extracellular enveloped virus (EV) orthopoxvirus protein.
- Orthopoxvirus is a genus of viruses in the family Poxviridae and subfamily Chordopoxvirinae. Vertebrates, including mammals and humans, and arthropods serve as natural hosts. There are at least 12 species in this genus, four of which can infect humans (smallpox virus, monkeypox virus, vaccina virus, and cowpox virus). Smallpox spread between subjects is typically respiratory, although contact with infectious skin lesions or scabs has been reported. As noted above, smallpox was eradicated in the late 1970s.
- MV intracellular mature virus
- EV extracellular enveloped virus
- MV is an enveloped virus with many surface proteins required for infectivity (e.g., LIR, A27L, A17L, D8L and H3L)
- EV has an additional membrane surrounding the MV particle and another set of unique membrane proteins (e.g., A33R and B5R). Both EV and MV are important in viral acquisition and spread.
- RNA e.g., mRNA
- the vaccine comprises RNA (e.g., mRNA) polynucleotides encoding at least two of the following antigens: A35R, B6R, M1R, and A29L.
- the vaccine comprises RNA (e.g., mRNA) polynucleotides encoding at least three of the following antigens: A35R, B6R, M1R, and A29L.
- the vaccine comprises RNA (e.g., mRNA) polynucleotides encoding the following antigens: A35R, B6R, M1R, and A29L.
- the vaccine comprises RNA (e.g., mRNA) polynucleotides encoding at least on MV antigen and at least one EV antigen.
- kits for administering the vaccines are also provided herein.
- methods of producing the vaccines may be used to induce a balanced immune response, comprising both cellular and humoral immunity, without many of the risks associated with DNA vaccination.
- a vaccine optionally referred to herein as a multivalent vaccine, can be administered to seropositive or seronegative subjects.
- a subject may be na ⁇ ve and not have antibodies that react with at least one of the antigenic polypeptides of the vaccine, or may have preexisting antibodies to at least one of antigens of the vaccine because they have previously had an infection with the orthopoxvirus or may have previously been administered a dose of a vaccine (e.g., an mRNA vaccine) that induces antibodies against the orthopoxvirus.
- a subject may have preexisting antibodies to all of the antigens of the vaccine.
- Antigens Antigens are proteins capable of inducing an immune response (e.g., causing an immune system to produce antibodies against the antigens).
- the vaccines of the present disclosure provide a unique advantage over traditional protein-based vaccination approaches in which protein antigens are purified or produced in vitro, e.g., recombinant protein production technologies.
- the vaccines of the present disclosure feature mRNA encoding the desired antigens, which when introduced into the body, i.e., administered to a mammalian subject (for example a human) in vivo, cause the cells of the body to express the desired antigens.
- the vaccines of the present disclosure feature mRNA encoding the desired viral membrane (surface) antigens, e.g., glycoprotein antigens, which when introduced into the body, i.e., administered to a mammalian subject (for example a human) in vivo, cause the cells of the body to express the desired peptides in a native fold and, optionally with human glycosylation patterns.
- a vaccine encoding the viral surface antigen from a series of pathogenic viruses all presenting the properly folded and, optionally, glycosylated viral antigens in the same manner as if it were generated during an actual infection.
- mRNA vaccines thus offer the best vehicle for making vaccines to viruses one can produce short of using an attenuated virus, but without the associated risks.
- the mRNAs are encapsulated in lipid nanoparticles (LNPs).
- LNPs lipid nanoparticles
- the mRNAs Upon delivery and uptake by cells of the body, the mRNAs are translated in the cytosol and protein or glycoprotein antigens are folded and processed by the host cell machinery.
- the protein and/or glycoprotein antigens are presented and elicit an adaptive humoral and cellular immune response.
- Neutralizing antibodies are directed against the expressed viral antigens and hence these viral protein antigens are considered the most relevant target antigens for vaccine development.
- neutralizing antibodies are generally directed to the viral surface proteins which are responsible for binding to the cell and when blocked by a specific antibody, the virus is neutralized.
- use of the term “antigen” encompasses immunogenic viral surface proteins and immunogenic fragments (an immunogenic fragment that induces (or is capable of inducing) an immune response to a (at least one) orthopoxvirus), unless otherwise stated.
- the antigen is a naturally occurring antigen (e.g., the antigenic polypeptide encodes a naturally occurring antigen).
- at least one antigenic polypeptide is a non-naturally occurring antigen or an engineered version of the protein or glycoprotein antigen for use in a vaccine.
- At least one of the antigenic polypeptides is a stabilized version of a naturally occurring antigen.
- other modifications are engineered into the viral surface protein, such as deletion of cytoplasmic tails or mutations to facilitate protein processing or conformational stability.
- protein encompasses glycoproteins, proteins, peptides and fragments thereof and the term “antigen” encompasses antigenic portions of such molecules that provoke an immune response.
- the term “antigen” includes viral surface proteins, e.g., ectodomains, fragments of viral proteins (e.g., glycoproteins) and designed and or mutated versions of viral proteins (e.g., glycoproteins) derived from orthopoxviruses.
- the antigens are from the suspected multi-country outbreak in May of 2022 (first confirmed case was in Portugal).
- the 2022 genome also comprises clusters with 2018-19 monkeypox virus (MPXV) sequences from the UK, Singapore, and Israel.
- the antigens are from the Zaire_I_96 reference strain.
- the antigens are from the Zaire_79 strain.
- the antigens are from the Zaire_I_96, Zaire_79 strain, the MPXV Singapore 2019 strain, and the MPXV Portugal 2022 strain, or any combination thereof.
- the mRNA vaccines of the instant invention comprise mRNAs encoding MV and/or EV antigens of an orthopoxvirus (e.g., monkeypox virus or smallpox virus).
- the mRNA vaccine comprises mRNAs encoding an MV antigen (e.g M1R or A29L).
- the mRNA vaccine comprises mRNA encoding an EV antigen (e.g., A35R or B6R).
- the mRNAs encode at least one MV antigen and at least one EV antigen. In some embodiments, the mRNA encoding at least one MV antigen and the mRNA encoding at least one EV antigen are present in the vaccine at a 1:1 ratio, a 2:1 ratio, a 3:1 ratio, a 4:1 ratio, a 1:4 ratio, a 1:3 ratio, or a 1:2 ratio.
- the antigen is an M1R protein (an ortholog of vaccinia L1R). The L1R protein is essential for vaccinia virus (VACV) replication and interacts with the entry-fusion complex (EFC). It is found on the surface of the mature virus (MV).
- the antigen is a B6R protein (an ortholog of vaccina B5R). The protein is found on the surface of the EV. In some embodiments, the antigen is an A35R protein (an ortholog of vaccinia A33R). The protein is found on the surface of the EV.
- A33 is a type II integral membrane protein that forms disulfide-bonded homodimers or heteromultimers (Roper, Payne, and Moss, 1996). It coordinates the incorporation of A36 into IEV membranes and subsequently, the production of actin tails (Wolffe, Weisberg, and Moss, 2001). Deletion of A33R causes a small plaque phenotype.
- the antigen is an A29L protein (an ortholog of A27L).
- the A27L protein is present on the surface of the mature virus, and it mediates vaccinia virus interaction with cell surface heparan sulfate.
- the antigen is any one of the antigens provided herein. In some embodiments, the antigen is 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or identical to any one of SEQ ID NOs: 7-34. In each embodiment or aspect of the invention, it is understood that the featured vaccines include the mRNAs encapsulated within LNPs.
- compositions of the present disclosure comprise a (at least one) messenger RNA (mRNA) having an open reading frame (ORF) encoding an orthopoxvirus (e.g., smallpox or monkeypox) antigen.
- mRNA messenger RNA
- ORF open reading frame
- orthopoxvirus e.g., smallpox or monkeypox
- the ORF is 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or identical to any one of SEQ ID NOs: 35-62.
- the mRNA further comprises a 5 ⁇ UTR, 3 ⁇ UTR, a poly(A) tail and/or a 5 ⁇ cap analog.
- the first, second and/or third mRNA polynucleotides in the composition differ in length from one another by at least 100 nucleotides (e.g., 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more nucleotides).
- the orthopoxvirus virus vaccine of the present disclosure may include any 5′ untranslated region (UTR) and/or any 3′ UTR.
- UTR sequences include SEQ ID NOs: 1-4 and 92-93; however, other UTR sequences may be used or exchanged for any of the UTR sequences described herein.
- a 5' UTR of the present disclosure comprises a sequence selected from SEQ ID NO: 1 (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC), SEQ ID NO: 2 (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCC ACC), or SEQ ID NO.92 (GGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCUCGCAAC UAGCAAGCUUUUGUUCUCGCC).
- a 3' UTR of the present disclosure comprises a sequence selected from SEQ ID NO: 3 (UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC), SEQ ID NO: 4 (UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC) or SEQ ID NO.: 93 (UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC).
- UTRs may also be omitted from the RNA polynucleotides provided herein.
- Nucleic acids comprise a polymer of nucleotides (nucleotide monomers). Thus, nucleic acids are also referred to as polynucleotides.
- Nucleic acids may be or may include, for example, deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a ⁇ -D-ribo configuration, ⁇ -LNA having an ⁇ -L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino- ⁇ -LNA having a 2′- amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) and/or chimeras and/or combinations thereof.
- DNAs deoxyribonucleic acids
- RNAs ribonucleic acids
- TAAs glycol nucleic acids
- PNAs peptide nucle
- Messenger RNA is any RNA that encodes a (at least one) protein (a naturally- occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo.
- mRNA messenger RNA
- nucleic acid sequences set forth in the instant application may recite “T”s in a representative DNA sequence but where the sequence represents mRNA, the “T”s would be substituted for “U”s.
- any of the DNAs disclosed and identified by a particular sequence identification number herein also disclose the corresponding mRNA sequence complementary to the DNA, where each “T” of the DNA sequence is substituted with “U.”
- An open reading frame is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)) and ending with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA).
- An ORF typically encodes a protein.
- compositions of the present disclosure include RNA that encodes virus antigens (e.g., MV and/or EV antigens) and structurally altered variants representing a plurality of virus antigens.
- virus antigens e.g., MV and/or EV antigens
- structurally altered variants representing a plurality of virus antigens.
- Antigenic variants or structurally altered variants refers to molecules that differ in their amino acid sequence from a wild-type (naturally occurring), native, or reference protein sequence.
- the antigen/ structurally altered variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants possess at least 50% identity to a wild-type, native or reference sequence. In some embodiments, variants share at least 80%, or at least 90% identity with a wild-type, native, or reference sequence.
- Variant antigens/polypeptides encoded by nucleic acids of the disclosure may contain amino acid changes that confer any of a number of desirable properties, e.g., that enhance their immunogenicity, vary the breadth of their immunogenicity, i.e.
- variant antigens/polypeptides can be made using routine mutagenesis techniques and assayed as appropriate to determine whether they possess the desired property. Assays to determine expression levels and immunogenicity are well known in the art and exemplary such assays are set forth in the Examples section. Similarly, PK/PD properties of a protein variant can be measured using art recognized techniques, e.g., by determining expression of antigens in a vaccinated subject over time and/or by looking at the durability of the induced immune response.
- a composition comprises an RNA or an RNA ORF that comprises a nucleotide sequence of any one of the sequences provided herein, or comprises a nucleotide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of a wild-type (naturally occurring) or variant antigen.
- identity refers to a relationship between the sequences of two or more polypeptides (e.g. antigens) or polynucleotides (nucleic acids), as determined by comparing the sequences. Identity also refers to the degree of sequence relatedness between or among sequences as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related antigens or nucleic acids can be readily calculated by known methods.
- Percent (%) identity as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.
- variants of a particular polynucleotide or polypeptide have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.
- Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res.25:3389-3402).
- Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, T.F.
- polypeptide sequences encoding proteins or glycoproteins containing substitutions, insertions and/or additions, deletions, and covalent modifications with respect to reference sequences, in particular the polypeptide (e.g., antigen) sequences disclosed herein, are included within the scope of this disclosure.
- sequence tags or amino acids such as one or more lysines, can be added to peptide sequences (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide detection, purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation.
- amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences.
- Certain amino acids e.g., C-terminal or N-terminal residues
- sequences for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (such as, e.g., foldon regions) and the like may be substituted with alternative sequences that achieve the same or a similar function.
- cavities in the core of proteins can be filled to improve stability, e.g., by introducing larger amino acids.
- buried hydrogen bond networks may be replaced with hydrophobic resides to improve stability.
- glycosylation sites may be removed and replaced with appropriate residues.
- sequences are readily identifiable to one of skill in the art. It should also be understood that some of the sequences provided herein contain sequence tags or terminal peptide sequences (e.g., at the N-terminal or C-terminal ends) that may be deleted, for example, prior to use in the preparation of an mRNA vaccine.
- protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of orthopoxvirus antigens of interest.
- any protein fragment meaning a polypeptide sequence at least one amino acid residue shorter than a reference antigen sequence but otherwise identical
- the fragment is immunogenic and confers a protective immune response to an orthopoxvirus (e.g., smallpox or monkeypox).
- an orthopoxvirus e.g., smallpox or monkeypox
- a structurally altered variant includes an antigen that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations with respect to a reference antigen.
- Antigens/antigenic polypeptides can range in length from about 4, 6, or 8 amino acids to full length proteins.
- Stabilizing Elements Naturally-occurring eukaryotic mRNA molecules can contain stabilizing elements, including, but not limited to untranslated regions (UTR) at their 5′-end (5′ UTR) and/or at their 3′-end (3′ UTR), in addition to other structural features, such as a 5′-cap structure or a 3′-poly(A) tail. Both the 5′ UTR and the 3′ UTR are typically transcribed from the genomic DNA and are elements of the premature mRNA.
- UTR untranslated regions
- a composition includes an RNA polynucleotide having an open reading frame encoding at least one antigenic polypeptide having at least one modification, at least one 5′ terminal cap, and is formulated within a lipid nanoparticle.5′-capping of polynucleotides may be completed concomitantly during the in vitro-transcription reaction using the following chemical RNA cap analogs to generate the 5′-guanosine cap structure according to manufacturer protocols: 3 ⁇ -O-Me-m7G(5')ppp(5') G [the ARCA cap];G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G
- Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2′-O methyl-transferase to generate: m7G(5')ppp(5')G-2′-O-methyl.
- Cap 2 structure may be generated from the Cap 1 structure followed by the 2′-O-methylation of the 5′-antepenultimate nucleotide using a 2′-O methyl-transferase.
- Cap 3 structure may be generated from the Cap 2 structure followed by the 2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-O methyl-transferase.
- Enzymes may be derived from a recombinant source.
- the 3′-poly(A) tail is typically a stretch of adenine nucleotides added to the 3′-end of the transcribed mRNA. It can, in some instances, comprise up to about 400 adenine nucleotides.
- the length of the 3′-poly(A) tail may be an essential element with respect to the stability of the individual mRNA.
- the vaccine e.g., multivalent RNA composition
- a composition includes a stabilizing element. Stabilizing elements may include for instance a histone stem-loop.
- a stem-loop binding protein (SLBP), a 32 kDa protein has been identified. It is associated with the histone stem-loop at the 3'-end of the histone messages in both the nucleus and the cytoplasm. Its expression level is regulated by the cell cycle; it peaks during the S-phase, when histone mRNA levels are also elevated. The protein has been shown to be essential for efficient 3'-end processing of histone pre-mRNA by the U7 snRNP. SLBP continues to be associated with the stem-loop after processing, and then stimulates the translation of mature histone mRNAs into histone proteins in the cytoplasm.
- SLBP stem-loop binding protein
- RNA binding domain of SLBP is conserved through metazoa and protozoa; its binding to the histone stem-loop depends on the structure of the loop.
- the minimum binding site includes at least three nucleotides 5’ and two nucleotides 3′ relative to the stem-loop.
- an mRNA includes a coding region, at least one histone stem- loop, and optionally, a poly(A) sequence or polyadenylation signal.
- the poly(A) sequence or polyadenylation signal generally should enhance the expression level of the encoded protein.
- the encoded protein in some embodiments, is not a histone protein, a reporter protein (e.g.
- an mRNA includes the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop, even though both represent alternative mechanisms in nature, acts synergistically to increase the protein expression beyond the level observed with either of the individual elements.
- an mRNA does not include a histone downstream element (HDE).
- Histone downstream element includes a purine-rich polynucleotide stretch of approximately 15 to 20 nucleotides 3′ of naturally occurring stem-loops, representing the binding site for the U7 snRNA, which is involved in processing of histone pre-mRNA into mature histone mRNA.
- the nucleic acid does not include an intron.
- an mRNA may or may not contain an enhancer and/or promoter sequence, which may be modified or unmodified or which may be activated or inactivated.
- the histone stem-loop is generally derived from histone genes and includes an intramolecular base pairing of two neighbored partially or entirely reverse complementary sequences separated by a spacer, consisting of a short sequence, which forms the loop of the structure.
- the unpaired loop region is typically unable to base pair with either of the stem loop elements. It occurs more often in RNA, as is a key component of many RNA secondary structures but may be present in single- stranded DNA as well. Stability of the stem-loop structure generally depends on the length, number of mismatches or bulges, and base composition of the paired region.
- wobble base pairing (non-Watson-Crick base pairing) may result.
- the at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides.
- an mRNA has one or more AU-rich sequences removed. These sequences, sometimes referred to as AURES are destabilizing sequences found in the 3’UTR. The AURES may be removed from the RNA vaccines. Alternatively, the AURES may remain in the RNA vaccine.
- Signal Peptides In some embodiments, a composition comprises an mRNA having an ORF that encodes a signal peptide fused to an orthopoxvirus antigen.
- Signal peptides comprising the N-terminal 15- 60 amino acids of proteins, are typically needed for the translocation across the membrane on the secretory pathway and, thus, universally control the entry of most proteins both in eukaryotes and prokaryotes to the secretory pathway.
- the signal peptide of a nascent precursor protein directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it for processing.
- ER processing produces mature proteins, wherein the signal peptide is cleaved from precursor proteins, typically by an ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor.
- a signal peptide may also facilitate the targeting of the protein to the cell membrane.
- a signal peptide may have a length of 15-60 amino acids.
- a signal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids.
- a signal peptide has a length of 20-60, 25-60, 30-60, 35- 60, 40-60, 45- 60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20- 40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15-20 amino acids.
- a composition of the present disclosure includes an mRNA encoding an antigenic fusion protein.
- the encoded antigen or antigens may include two or more proteins (e.g., protein and/or protein fragment) joined together.
- the protein to which a protein antigen is fused does not promote a strong immune response to itself, but rather to the orthopoxvirus antigen.
- Antigenic fusion proteins retain the functional property from each original protein.
- scaffold moieties encode fusion proteins that comprise orthopoxvirus antigens linked to scaffold moieties.
- scaffold moieties impart desired properties to an antigen encoded by a nucleic acid of the disclosure.
- scaffold proteins may improve the immunogenicity of an antigen, e.g., by altering the structure of the antigen, altering the uptake and processing of the antigen, and/or causing the antigen to bind to a binding partner.
- the scaffold moiety is protein that can self-assemble into protein nanoparticles that are highly symmetric, stable, and structurally organized, with diameters of 10– 150 nm, a highly suitable size range for optimal interactions with various cells of the immune system.
- viral proteins or virus-like particles can be used to form stable nanoparticle structures. Examples of such viral proteins are known in the art.
- the scaffold moiety is a hepatitis B surface antigen (HBsAg). HBsAg forms spherical particles with an average diameter of ⁇ 22 nm and which lacked nucleic acid and hence are non-infectious (Lopez-Sagaseta, J. et al.
- the scaffold moiety is a hepatitis B core antigen (HBcAg) self-assembles into particles of 24–31 nm diameter, which resembled the viral cores obtained from HBV-infected human liver.
- HBcAg produced in self-assembles into two classes of differently sized nanoparticles of 300 ⁇ and 360 ⁇ diameter, corresponding to 180 or 240 protomers.
- the orthopoxvirus antigen is fused to HBsAG or HBcAG to facilitate self-assembly of nanoparticles displaying the orthopoxvirus antigen.
- bacterial protein platforms may be used.
- Non-limiting examples of these self-assembling proteins include ferritin, lumazine and encapsulin.
- Ferritin is a protein whose main function is intracellular iron storage.
- Ferritin is made of 24 subunits, each composed of a four-alpha-helix bundle, that self-assemble in a quaternary structure with octahedral symmetry (Cho K.J. et al. J Mol Biol.2009;390:83–98).
- Several high- resolution structures of ferritin have been determined, confirming that Helicobacter pylori ferritin is made of 24 identical protomers, whereas in animals, there are ferritin light and heavy chains that can assemble alone or combine with different ratios into particles of 24 subunits (Granier T.
- Ferritin self-assembles into nanoparticles with robust thermal and chemical stability.
- the ferritin nanoparticle is well-suited to carry and expose antigens.
- Lumazine synthase (LS) is also well-suited as a nanoparticle platform for antigen display.
- LS which is responsible for the penultimate catalytic step in the biosynthesis of riboflavin, is an enzyme present in a broad variety of organisms, including archaea, bacteria, fungi, plants, and eubacteria (Weber S.E. Flavins and Flavoproteins.
- the LS monomer is 150 amino acids long and consists of beta-sheets along with tandem alpha-helices flanking its sides.
- a number of different quaternary structures have been reported for LS, illustrating its morphological versatility: from homopentamers up to symmetrical assemblies of 12 pentamers forming capsids of 150 ⁇ diameter. Even LS cages of more than 100 subunits have been described (Zhang X. et al. J Mol Biol.2006;362:753–770).
- Encapsulin a novel protein cage nanoparticle isolated from thermophile Thermotoga maritima, may also be used as a platform to present antigens on the surface of self-assembling nanoparticles.
- an RNA of the present disclosure encodes an orthopoxvirus antigen fused to a foldon domain.
- the foldon domain may be, for example, obtained from bacteriophage T4 fibritin (see, e.g., Tao Y, et al. Structure.1997 Jun 15; 5(6):789-98).
- the mRNAs of the disclosure encode more than one polypeptide, referred to herein as fusion proteins.
- the mRNA further encodes a linker located between at least one or each domain of the fusion protein.
- the linker can be, for example, a cleavable linker or protease-sensitive linker.
- the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, E2A linker, and combinations thereof.
- This family of self-cleaving peptide linkers, referred to as 2A peptides has been described in the art (see for example, Kim, J.H. et al.
- the linker is an F2A linker. In some embodiments, the linker is a GGGS (SEQ ID NO: 63) linker. In some embodiments, the fusion protein contains three domains with intervening linkers, having the structure: domain-linker-domain-linker-domain. Cleavable linkers known in the art may be used in connection with the disclosure. Exemplary such linkers include: F2A linkers,T2A linkers, P2A linkers, E2A linkers (See, e.g., WO2017127750).
- an ORF encoding an antigen of the disclosure is codon optimized. Codon optimization methods are known in the art. For example, an ORF of any one or more of the sequences provided herein may be codon optimized.
- Codon optimization may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide.
- encoded protein e.g., glycosylation sites
- add, remove or shuffle protein domains add or delete restriction sites
- modify ribosome binding sites and mRNA degradation sites adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide.
- Codon optimization tools, algorithms and services are known in the art – non- limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods.
- the open reading frame (ORF) sequence is optimized using optimization algorithms.
- a codon optimized sequence shares less than 95% sequence identity to a naturally-occurring or wild-type sequence ORF (e.g., a naturally-occurring or wild- type mRNA sequence encoding an orthopoxvirus antigen).
- a codon optimized sequence shares less than 90% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding an orthopoxvirus antigen). In some embodiments, a codon optimized sequence shares less than 85% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding an orthopoxvirus antigen). In some embodiments, a codon optimized sequence shares less than 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding an orthopoxvirus antigen).
- a codon optimized sequence shares less than 75% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding an orthopoxvirus antigen). In some embodiments, a codon optimized sequence shares between 65% and 85% (e.g., between about 67% and about 85% or between about 67% and about 80%) sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding an orthopoxvirus antigen).
- a codon optimized sequence shares between 65% and 75% or about 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding an orthopoxvirus antigen).
- a codon-optimized sequence encodes an antigen that is as immunogenic as, or more immunogenic than (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% more), than an orthopoxvirus antigen encoded by a non-codon-optimized sequence.
- the modified mRNAs When transfected into mammalian host cells, the modified mRNAs have a stability of between 12-18 hours, or greater than 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours and are capable of being expressed by the mammalian host cells.
- a codon optimized RNA may be one in which the levels of G/C are enhanced.
- the G/C-content of nucleic acid molecules (e.g., mRNA) may influence the stability of the RNA.
- RNA having an increased amount of guanine (G) and/or cytosine (C) residues may be functionally more stable than RNA containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides.
- WO02/098443 discloses a pharmaceutical composition containing an mRNA stabilized by sequence modifications in the translated region. Due to the degeneracy of the genetic code, the modifications work by substituting existing codons for those that promote greater RNA stability without changing the resulting amino acid. The approach is limited to coding regions of the RNA.
- an mRNA is not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine.
- nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g. A, G, C, or U).
- nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g. dA, dG, dC, or dT).
- compositions of the present disclosure comprise, in some embodiments, an RNA having an open reading frame encoding an orthopoxvirus antigen, wherein the nucleic acid comprises nucleotides and/or nucleosides that can be standard (unmodified) or modified as is known in the art.
- nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides.
- modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides.
- Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art.
- a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.
- a non-naturally occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos.
- nucleic acids of the disclosure can comprise standard nucleotides and nucleosides, naturally- occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof.
- Nucleic acids of the disclosure e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids
- in some embodiments comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides.
- a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides.
- a modified RNA nucleic acid e.g., a modified mRNA nucleic acid
- introduced to a cell or organism exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.
- a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.
- Nucleic acids e.g., RNA nucleic acids, such as mRNA nucleic acids
- nucleic acid e.g., RNA nucleic acids, such as mRNA nucleic acids.
- a “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”).
- nucleotide refers to a nucleoside, including a phosphate group.
- Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides.
- Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides.
- Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures, such as, for example, in those nucleic acids having at least one chemical modification.
- non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil.
- modified nucleobases in nucleic acids comprise 1-methyl-pseudouridine (m1 ⁇ ), 1-ethyl-pseudouridine (e1 ⁇ ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine ( ⁇ ).
- modified nucleobases in nucleic acids comprise 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, and/or 5-methoxy cytidine.
- the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.
- a mRNA of the disclosure comprises 1-methyl-pseudouridine (m1 ⁇ ) substitutions at one or more or all uridine positions of the nucleic acid.
- a mRNA of the disclosure comprises 1-methyl-pseudouridine (m1 ⁇ ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.
- a mRNA of the disclosure comprises pseudouridine ( ⁇ ) substitutions at one or more or all uridine positions of the nucleic acid.
- a mRNA of the disclosure comprises pseudouridine ( ⁇ ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.
- a mRNA of the disclosure comprises uridine at one or more or all uridine positions of the nucleic acid.
- mRNAs are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification.
- a nucleic acid can be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine.
- a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.
- the nucleic acids of the present disclosure may be partially or fully modified along the entire length of the molecule.
- one or more or all or a given type of nucleotide may be uniformly modified in a nucleic acid of the disclosure, or in a predetermined sequence region thereof (e.g., in the mRNA including or excluding the poly(A) tail).
- nucleotides X in a nucleic acid of the present disclosure are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
- the nucleic acid may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to
- the mRNAs may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides.
- the nucleic acids may contain a modified pyrimidine such as a modified uracil or cytosine.
- At least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the nucleic acid is replaced with a modified uracil (e.g., a 5-substituted uracil).
- the modified uracil can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
- cytosine in the nucleic acid is replaced with a modified cytosine (e.g., a 5-substituted cytosine).
- the modified cytosine can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
- Untranslated Regions UTRs
- the mRNAs of the present disclosure may comprise one or more regions or parts which act or function as an untranslated region.
- the nucleic may comprise one or more of these untranslated regions (UTRs). Wild-type untranslated regions of a nucleic acid are transcribed but not translated. In mRNA, the 5′ UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas the 3′ UTR starts immediately following the stop codon and continues until the transcriptional termination signal.
- UTRs untranslated regions
- a variety of 5’UTR and 3’UTR sequences are known and available in the art.
- a 5 ⁇ UTR is region of an mRNA that is directly upstream (5 ⁇ ) from the start codon (the first codon of an mRNA transcript translated by a ribosome).
- a 5 ⁇ UTR does not encode a protein (is non-coding).
- Natural 5′UTRs have features that play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes.
- a 5’ UTR is a heterologous UTR, i.e., is a UTR found in nature associated with a different ORF.
- a 5’ UTR is a synthetic UTR, i.e., does not occur in nature.
- Synthetic UTRs include UTRs that have been mutated to improve their properties, e.g., which increase gene expression as well as those which are completely synthetic.
- Exemplary 5’ UTRs include Xenopus or human derived a-globin or b- globin (8278063; 9012219), human cytochrome b-245 a polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus (US8278063, 9012219).
- CMV immediate-early 1 (IE1) gene (US20140206753, WO2013/185069), the sequence GGGAUCCUACC (SEQ ID NO: 6) (WO2014144196) may also be used.
- 5' UTR of a TOP gene is a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract) (e.g., WO/2015101414, WO2015101415, WO/2015/062738, WO2015024667, WO2015024667; 5' UTR element derived from ribosomal protein Large 32 (L32) gene (WO/2015101414, WO2015101415, WO/2015/062738), 5' UTR element derived from the 5'UTR of an hydroxysteroid (17- ⁇ ) dehydrogenase 4 gene (HSD17B4) (WO2015024667), or a 5' UTR element derived from the 5' UTR of ATP5A1 (WO2015024667) can be used.
- L32 ribosomal protein Large 32
- HSD17B4 hydroxysteroid
- HSD17B4 hydroxysteroid
- WO2015024667 or a 5' UTR element
- an internal ribosome entry site is used instead of a 5' UTR.
- a 5' UTR of the present disclosure comprises a sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2.
- a 3 ⁇ UTR is region of an mRNA that is directly downstream (3 ⁇ ) from the stop codon (the codon of an mRNA transcript that signals a termination of translation).
- a 3 ⁇ UTR does not encode a protein (is non-coding).
- Natural or wild type 3′ UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover.
- AU rich elements can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class.
- AREs 3′ UTR AU rich elements
- AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein.
- Transfection experiments can be conducted in relevant cell lines, using nucleic acids of the disclosure and protein production can be assayed at various time points post-transfection.
- cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.
- 5’UTRs that are heterologous or synthetic may be used with any desired 3’ UTR sequence.
- a heterologous 5’UTR may be used with a synthetic 3’UTR with a heterologous 3’ UTR.
- Non-UTR sequences may also be used as regions or subregions within a nucleic acid.
- introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure. Incorporation of intronic sequences may increase protein production as well as nucleic acid levels. Combinations of features may be included in flanking regions and may be contained within other features.
- the ORF may be flanked by a 5' UTR which may contain a strong Kozak translational initiation signal and/or a 3' UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail.
- 5′ UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes such as the 5′ UTRs described in US Patent Application Publication No.20100293625 and PCT/US2014/069155, herein incorporated by reference in its entirety. It should be understood that any UTR from any gene may be incorporated into the regions of a nucleic acid. Furthermore, multiple wild-type UTRs of any known gene may be utilized.
- UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5′ or 3′ UTR may be inverted, shortened, lengthened, made with one or more other 5′ UTRs or 3′ UTRs.
- altered as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence.
- a 3′ UTR or 5′ UTR may be altered relative to a wild-type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3′ or 5′) comprise a variant UTR.
- a double, triple or quadruple UTR such as a 5′ UTR or 3′ UTR may be used.
- a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series.
- a double beta-globin 3′ UTR may be used as described in US Patent publication 20100129877, the contents of which are incorporated herein by reference in its entirety. It is also within the scope of the present disclosure to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level. In some embodiments, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature or property.
- polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development.
- the UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide.
- a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.
- the untranslated region may also include translation enhancer elements (TEE).
- TEE translation enhancer elements
- the TEE may include those described in US Application No.20090226470, herein incorporated by reference in its entirety, and those known in the art.
- RNA transcript e.g., mRNA transcript
- a DNA template e.g., a first input DNA and a second input DNA
- a RNA polymerase e.g., a T7 RNA polymerase, a T7 RNA polymerase variant, etc.
- IVT in vitro transcription
- IVT conditions typically require a purified linear DNA template containing a promoter, nucleoside triphosphates, a buffer system that includes dithiothreitol (DTT) and magnesium ions, and a RNA polymerase.
- DTT dithiothreitol
- RNA polymerase a RNA polymerase that includes dithiothreitol (DTT) and magnesium ions.
- Typical IVT reactions are performed by incubating a DNA template with a RNA polymerase and nucleoside triphosphates, including GTP, ATP, CTP, and UTP (or nucleotide analogs) in a transcription buffer.
- a RNA transcript having a 5 ⁇ terminal guanosine triphosphate is produced from this reaction.
- a wild-type T7 polymerase is used in an IVT reaction.
- a modified or mutant T7 polymerase is used in an IVT reaction.
- a T7 RNA polymerase variant comprises an amino acid sequence that shares at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity with a wild-type T7 (WT T7) polymerase.
- WT T7 wild-type T7
- the T7 polymerase variant is a T7 polymerase variant described by International Application Publication Number WO2019/036682 or WO2020/172239, the entire contents of each of which are incorporated herein by reference.
- the RNA polymerase (e.g., T7 RNA polymerase or T7 RNA polymerase variant) is present in a reaction (e.g., an IVT reaction) at a concentration of 0.01 mg/ml to 1 mg/ml.
- a reaction e.g., an IVT reaction
- the RNA polymerase may be present in a reaction at a concentration of 0.01 mg/mL, 0.05 mg/ml, 0.1 mg/ml, 0.5 mg/ml or 1.0 mg/ml.
- the input deoxyribonucleic acid (DNA) serves as a nucleic acid template for RNA polymerase.
- a DNA template may include a polynucleotide encoding a polypeptide of interest (e.g., an antigenic polypeptide).
- a DNA template in some embodiments, includes a RNA polymerase promoter (e.g., a T7 RNA polymerase promoter) located 5’ from and operably linked to polynucleotide encoding a polypeptide of interest.
- a DNA template may also include a nucleotide sequence encoding a polyadenylation (polyA) tail located at the 3’ end of the gene of interest.
- an input DNA comprises plasmid DNA (pDNA).
- pDNA plasmid DNA
- plasmid DNA or “pDNA” refers to an extrachromosomal DNA molecule that is physically separated from chromosomal DNA in a cell and can replicate independently.
- plasmid DNA is isolated from a cell (e.g., as a plasmid DNA preparation).
- plasmid DNA comprises an origin of replication, which may contain one or more heterologous nucleic acids, for example nucleic acids encoding therapeutic proteins that may serve as a template for RNA polymerase.
- Plasmid DNA may be circularized or linear (e.g., plasmid DNA that has been linearized by a restriction enzyme digest).
- Multivalent mRNA constructs are typically produced by transcribing one mRNA product at a time, purifying each mRNA product, and then mixing the purified mRNA products together prior to formulation. This type of process incurs significant time and monetary investment especially at the Good Manufacturing Practice (GMP) scale.
- GMP Good Manufacturing Practice
- compositions comprising multivalent different RNAs (e.g., 2 or more different RNAs).
- methods of multivalent transcription disclosed herein involve selecting amounts of input DNA for IVT reactions that result in multivalent RNA compositions having higher purity than RNA compositions produced using previous methods.
- RNA polymerase e.g., RNA polymerase, nucleotide triphosphates (NTPs), etc.
- NTPs nucleotide triphosphates
- modifying input DNA amounts results in production of multivalent RNA compositions having increased purity (e.g., as measured by percentage of RNAs comprising polyA tails) relative to RNA compositions produced by previous methods.
- the disclosure provides a method for producing a multivalent RNA composition, the method comprising simultaneously in vitro transcribing at least two DNA molecules in a reaction mixture comprising: a first population of DNA molecules encoding a first RNA; a second population of DNA molecules encoding a second RNA that is different than the first RNA; and obtaining a multivalent RNA composition having a pre-defined ratio of the first RNA to the second RNA produced by the IVT.
- multivalent RNA composition refers to a composition comprising more than two different mRNAs.
- a multivalent RNA composition may comprise 2 or more different RNAs, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different RNAs.
- a multivalent RNA composition comprises more than 10 different RNAs.
- the term “different RNAs” refers to any RNA that is not the same as another RNA in a multivalent RNA composition.
- each input DNA e.g., population of input DNA molecules
- each input DNA is obtained from a different source (e.g., synthesized separately, for example in different cells or populations of cells).
- each input DNA is obtained from a different bacterial cell or population of bacterial cells.
- the first input DNA is produced in bacterial cell population A
- the second input DNA is produced in bacterial cell population B
- the third input DNA is produced in bacterial population C, where each of A, B, and C are not the same bacterial culture (e.g., co-cultured in the same container or plate).
- Methods of obtaining populations of input DNAs are known, for example as described by Sambrook, Joseph. Molecular Cloning : a Laboratory Manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2001. Some aspects comprise normalizing the amount of DNA used in the multivalent co-IVT reaction.
- the normalization is based on the molar mass of the input DNAs. In some embodiments, the normalization is based on the degradation rate of the input DNAs. In some embodiments, the normalization is based on the degradation rate of the resultant mRNAs (e.g., measured based upon polyA variants present in the reaction mixture, or T7 polymerase abortive transcripts or truncated transcripts). In some embodiments, the normalization is based on the nucleotide content (e.g., amount of A, G, C, U, or any combination thereof) of the input DNAs. In some embodiments, the normalization is based on the purity of the input DNAs.
- the normalization is based on the polyA-tailing efficiency of the input DNAs. In some embodiments, the normalization is based on the lengths of the input DNAs. In some embodiments, mRNA is at a pre-defined mRNA ratio, which may comprise a ratio between 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different RNAs (e.g., depending on the number of different RNAs in a composition). In some embodiments, a pre-defined ratio comprises a ratio between more than 10 RNAs. As used herein, a “pre-defined mRNA ratio” refers to the desired final ratio of RNA molecules in a multivalent RNA composition.
- RNA composition will depend upon the final peptide(s) or polypeptide product(s) encoded by the RNAs.
- a multivalent RNA mixture may comprise two RNAs (e.g., a RNA encoding a first antigen and a second antigen); in this instance the desired final ratio of RNA molecules may be 1 first antigen RNA:1 second antigen RNA.
- a multivalent RNA composition may comprise several (e.g., 3, 4, 5, 6, 7, 8, or more) RNAs encoding different antigenic peptides (e.g., for use as a vaccine); in that instance the desired ratio may comprise between 3 and 10 RNAs (e.g., a:b:c, a:b:c:d, a:b:c:d:e, a:b:c:d:e:f, a:b:c:d:e:f:g, a:b:c:d:e:f:g:h, a:b:c:d:e:f:g:h:i, a:b:c:d:e:f:g:h:i:j, etc., where each of a-j is a number between 1 and 10).
- the normalization is based on the lowest level present in the input DNAs (e.g., lowest molar mass, degradation rate (e.g., of the input DNA and/or output RNA), nucleotide content, purity, and/or polyA-tailing efficiency). In some embodiments, the normalization is based on the highest level present in the input DNAs (e.g., highest molar mass, degradation rate (e.g., of the input DNA and/or output RNA), nucleotide context, purity, and/or polyA-tailing efficiency).
- the normalization is based on the rate of RNA production of the input DNAs (e.g., the highest rate of RNA production of an input DNA or the lowest rate of RNA production of an input DNA in a reaction mixture).
- the disclosure relates to IVT methods in which the amount of input DNA (e.g., a first DNA or second DNA) is adjusted or normalized in order to improve production of multivalent RNA compositions having a pre-defined mRNA ratio of components.
- an IVT reaction mixture comprises 2 or more different input DNAs, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more different input DNAs.
- the IVT reaction comprises more than 15 different input DNAs.
- the term “different input DNAs” encompasses input DNAs that encode different RNAs, e.g., that have i) different lengths (whether or not the RNAs are identical over the entirety of the shorter of the two lengths), ii) different nucleotide sequences, iii) different chemical modification patterns, or iv) any combination of the foregoing.
- two or more of the input DNA molecules used in an IVT reaction encode mRNA molecules that have a different length (e.g., comprises a different number of nucleotides).
- the difference in length between two or more of the mRNA molecules encoded by different input DNA molecules in an IVT reaction mixture is greater than 70 nucleotides, 80 nucleotides, 90 nucleotides, or 100 nucleotides (e.g., two input DNAs in a composition encode mRNA molecules that are not are within 70, 80, 90, or 100 nucleotides in length of one another).
- the difference in length between two or more of the mRNA molecules encoded by different input DNA molecules is more than 100 nucleotides, for example 500 nucleotides, 1000 nucleotides, 1500 nucleotides, 2000 nucleotides, 3000 nucleotides, 4000 nucleotides, or more.
- the vaccine (e.g., multivalent RNA composition) is produced by combining a linearized first DNA molecule encoding the first mRNA polynucleotide, a linearized second DNA molecule encoding the second mRNA polynucleotide, and a linearized third DNA molecule encoding the third mRNA polynucleotide into a single reaction vessel, wherein the first DNA molecule, the second DNA molecule, and the third DNA molecule are obtained from different sources.
- the different sources are a first, second, and third bacterial cell culture and wherein the first, second and third bacterial cell culture are not co-cultured.
- the different sources are a first, second, and third bacterial cell culture and wherein the first, second and third bacterial cell culture are co-cultured.
- the amounts of the first, second and third DNA molecules present in the reaction mixture prior to the start of the in vitro transcription have been normalized.
- the linearized first DNA molecule, the linearized second DNA molecule and the linearized third DNA molecule are simultaneously in vitro transcribed to obtain the multivalent RNA composition.
- an in vitro transcription template encodes a 5′ untranslated (UTR) region, contains an open reading frame, and encodes a 3′ UTR and a poly(A) tail.
- UTR 5′ untranslated
- a “5′ untranslated region” refers to a region of an mRNA that is directly upstream (i.e., 5′) from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide.
- the 5’ UTR may comprise a promoter sequence. Such promoter sequences are known in the art. It should be understood that such promoter sequences will not be present in a vaccine of the disclosure.
- a “3′ untranslated region” refers to a region of an mRNA that is directly downstream (i.e., 3′) from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.
- An “open reading frame” is a continuous stretch of DNA beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a polypeptide.
- a “poly(A) tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3′), from the 3′ UTR that contains multiple, consecutive adenosine monophosphates.
- a poly(A) tail may contain 10 to 300 adenosine monophosphates.
- a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates.
- a poly(A) tail contains 50 to 250 adenosine monophosphates.
- the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, and/or export of the mRNA from the nucleus and translation.
- a nucleic acid includes 200 to 3,000 nucleotides.
- a nucleic acid may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides).
- An in vitro transcription system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase.
- NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein.
- the NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs.
- RNA polymerases or variants may be used in the method of the present disclosure.
- the polymerase may be selected from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and/or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids and/or modified nucleotides, including chemically modified nucleic acids and/or nucleotides. Some embodiments exclude the use of DNase. In some embodiments, the RNA transcript is capped via enzymatic capping.
- the RNA comprises 5' terminal cap, for example, 7mG(5’)ppp(5’)NlmpNp.
- mRNAs e.g., 2-15 mRNA polynucleotides each comprising a distinct open reading frame (ORF) encoding an orthopoxvirus antigenic polypeptide, wherein each mRNA polynucleotide comprises one or more non-coding sequences in an untranslated region (UTR) having unique identifier sequences or non-coding sequences.
- ORF open reading frame
- non-coding sequence refers to a sequence of a biological molecule (e.g., nucleic acid, protein, etc.) that when combined with the sequence another biological molecule serves to identify the other biological molecule.
- a non-coding sequence is a heterologous sequence that is incorporated within or appended to a sequence of a target biological molecule and utilized as a reference in order to identify a target molecule of interest.
- a non-coding sequence is a sequence of a nucleic acid (e.g., a heterologous or synthetic nucleic acid) that is incorporated within or appended to a target nucleic acid and utilized as a reference in order to identify the target nucleic acid.
- a non-coding sequence is of the formula (N)n.
- n is an integer in the range of 5 to 20, 5 to 10, 10 to 20, 7 to 20, or 7 to 30.
- n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more.
- N are each nucleotides that are independently selected from A, G, T, U, and C, or analogues thereof.
- nucleic acids e.g., mRNAs
- a target sequence of interest e.g., a coding sequence (e.g., that encodes therapeutic peptide or therapeutic protein)
- a coding sequence e.g., that encodes therapeutic peptide or therapeutic protein
- one or more in vitro transcribed mRNAs comprise one or more non-coding sequences in an untranslated region (UTR), such as a 5’ UTR or 3’ UTR. Inclusion of a non-coding sequence in the UTR of an mRNA prevents non-coding sequence from being translated into a peptide.
- a non-coding sequence is positioned in a 3’ UTR of an mRNA.
- the non-coding sequence is positioned upstream of the polyA tail of the mRNA. In some embodiments, the non-coding sequence is positioned downstream of (e.g., after) the polyA tail of the mRNA. In some embodiments, the non-coding sequence is positioned between the last codon of the ORF of the mRNA and the first “A” of the polyA tail of the mRNA. In some embodiments, a polynucleotide non-coding sequence positioned in a UTR comprises between 1 and 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides).
- UTR comprising a polynucleotide non-coding sequence further comprises one or more (e.g., 1, 2, 3, or more) RNAse cleavage sites, such as RNase H cleavage sites.
- each different RNA of a multivalent RNA composition comprises a different (e.g., unique) non-coding sequence.
- RNAs of a multivalent RNA composition are detected and/or purified according to the polynucleotide non- coding sequences of the RNAs.
- the mRNA non-coding sequences are used to identify the presence of mRNA or determine a relative ratio of different mRNAs in a sample (e.g., a reaction product or a drug product). In some embodiments, the mRNA non- coding sequences are detected using one or more of deep sequencing, PCR, and Sanger sequencing.
- Exemplary non-coding sequences include: AACGUGAU; AAACAUCG; ATGCCUAA; AGUGGUCA; ACCACUGU; ACAUUGGC; CAGAUCUG; CAUCAAGU; CGCUGAUC; ACAAGCUA; CUGUAGCC; AGUACAAG; AACAACCA; AACCGAGA; AACGCUUA; AAGACGGA; AAGGUACA; ACACAGAA; ACAGCAGA; ACCUCCAA; ACGCUCGA; ACGUAUCA; ACUAUGCA; AGAGUCAA; AGAUCGCA; AGCAGGAA; AGUCACUA; AUCCUGUA; AUUGAGGA; CAACCACA; GACUAGUA; CAAUGGAA; CACUUCGA; CAGCGUUA; CAUACCAA; CCAGUUCA; CCGAAGUA; ACAGUG; CGAUGU; UUAGGC; AUCACG; and UGACCA.
- the multivalent RNA composition is produced by a method comprising: (a) combining a linearized first DNA molecule encoding the first mRNA polynucleotide, a linearized second DNA molecule encoding the second mRNA polynucleotide, and a linearized third, fourth, fifth, sixth, seventh, eighth, ninth or tenth DNA molecule encoding the third, fourth, fifth, sixth, seventh, eighth, ninth or tenth mRNA polynucleotide into a single reaction vessel, wherein the first DNA molecule, the second DNA molecule, and the third, fourth, fifth, sixth, seventh, eighth, ninth or tenth DNA molecule are obtained from different sources; and (b) simultaneously in vitro transcribing the linearized first DNA molecule, the linearized second DNA molecule and the linearized third, fourth, fifth, sixth, seventh, eighth, ninth or tenth DNA molecule to obtain a multivalent RNA composition.
- the different sources may be bacterial cell cultures which may not be co-cultured.
- the amounts of the first, second and third, fourth, fifth, sixth, seventh, eighth, ninth or tenth DNA molecules present in the reaction mixture prior to the start of the IVT have been normalized.
- Chemical Synthesis Solid-phase chemical synthesis. Nucleic acids the present disclosure may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Solid-phase synthesis is useful in site-specific introduction of chemical modifications in the nucleic acid sequences. Liquid Phase Chemical Synthesis.
- nucleic acids of the present disclosure by the sequential addition of monomer building blocks may be carried out in a liquid phase.
- Combination of Synthetic Methods The synthetic methods discussed above each has its own advantages and limitations. Attempts have been conducted to combine these methods to overcome the limitations. Such combinations of methods are within the scope of the present disclosure.
- the use of solid-phase or liquid-phase chemical synthesis in combination with enzymatic ligation provides an efficient way to generate long chain nucleic acids that cannot be obtained by chemical synthesis alone. Ligation of Nucleic Acid Regions or Subregions Assembling nucleic acids by a ligase may also be used.
- DNA or RNA ligases promote intermolecular ligation of the 5’ and 3’ ends of polynucleotide chains through the formation of a phosphodiester bond.
- Nucleic acids such as chimeric polynucleotides and/or circular nucleic acids may be prepared by ligation of one or more regions or subregions. DNA fragments can be joined by a ligase catalyzed reaction to create recombinant DNA with different functions. Two oligodeoxynucleotides, one with a 5’ phosphoryl group and another with a free 3’ hydroxyl group, serve as substrates for a DNA ligase.
- nucleic acid clean-up may include, but is not limited to, nucleic acid clean-up, quality assurance and quality control. Clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).
- AGENCOURT® beads Beckman Coulter Genomics, Danvers, MA
- poly-T beads poly-T beads
- LNATM oligo-T capture probes EXIQON® Inc, Vedbaek, Denmark
- HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (
- purified when used in relation to a nucleic acid such as a “purified nucleic acid” refers to one that is separated from at least one contaminant.
- a “contaminant” is any substance that makes another unfit, impure or inferior.
- a purified nucleic acid e.g., DNA and RNA
- a quality assurance and/or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.
- the nucleic acids may be sequenced by methods including, but not limited to reverse-transcriptase-PCR. Quantification In some embodiments, the nucleic acids of the present disclosure may be quantified in exosomes or when derived from one or more bodily fluid.
- Bodily fluids include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood.
- CSF cerebrospinal fluid
- saliva aqueous humor
- amniotic fluid cerumen
- breast milk broncheoalveolar lavage fluid
- exosomes may be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.
- Assays may be performed using construct specific probes, cytometry, qRT-PCR, real- time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes may be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods.
- ELISA enzyme linked immunosorbent assay
- Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof. These methods afford the investigator the ability to monitor, in real time, the level of nucleic acids remaining or delivered. This is possible because the nucleic acids of the present disclosure, in some embodiments, differ from the endogenous forms due to the structural or chemical modifications. In some embodiments, the nucleic acid may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).
- UV/Vis ultraviolet visible spectroscopy
- a non-limiting example of a UV/Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, MA).
- the quantified nucleic acid may be analyzed in order to determine if the nucleic acid may be of proper size, check that no degradation of the nucleic acid has occurred.
- Degradation of the nucleic acid may be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC- HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
- LCMS liquid chromatography-mass spectrometry
- CE capillary electrophoresis
- CGE capillary gel electrophoresis
- LNPs Lipid Nanoparticles
- the mRNA of the disclosure is formulated in a lipid nanoparticle (LNP).
- Lipid nanoparticles typically comprise ionizable amino lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest.
- the lipid nanoparticles of the disclosure can be generated using components, compositions, and methods as are generally known in the art, see for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016/000129; PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/052117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491 all of which are incorporated by reference herein in their entirety.
- Vaccines of the present disclosure are typically formulated in lipid nanoparticles.
- the vaccines can be made, for example, using mixing processes such as microfluidics and T- junction mixing of two fluid streams, one of which contains the mRNA and the other has the lipid components.
- the vaccines are prepared by combining an ionizable amino lipid, a phospholipid (such as DOPE or DSPC), a PEG lipid (such as 1,2-dimyristoyl-OT- glycerol methoxypoly ethylene glycol, also known as PEG-DMG), and a structural lipid (such as cholesterol) in an alcohol (e.g., ethanol).
- a phospholipid such as DOPE or DSPC
- PEG lipid such as 1,2-dimyristoyl-OT- glycerol methoxypoly ethylene glycol, also known as PEG-DMG
- a structural lipid such as cholesterol
- the lipids may be combined to yield desired molar ratios and diluted with water and alcohol (e.g., ethanol) to a final lipid concentration of between about 5.5 mM and about 25 mM, for example.
- Vaccines including mRNA and a lipid component may be prepared, for example, by combining a lipid solution with an mRNA solution at lipid component to mRNA wt:wt ratios of between about 5:1 and about 50:1.
- the lipid solution may be rapidly injected using a microfluidic based system (e.g., NanoAssemblr) at flow rates between about 10 ml/min and about 18 ml/min, for example, into the mRNA solution to produce a suspension (e.g., with a water to alcohol ratio between about 1:1 and about 4:1).
- Vaccines can be processed by dialysis to remove the alcohol (e.g., ethanol) and achieve buffer exchange.
- Formulations may be dialyzed against phosphate buffered saline (PBS), pH 7.4, for example, at volumes greater than that of the primary product (e.g., using Slide-A-Lyzer cassettes (Thermo Fisher Scientific Inc., Rockford, IL)) with a molecular weight cutoff of 10 kD, for example.
- PBS phosphate buffered saline
- the forgoing exemplary method induces nanoprecipitation and particle formation.
- Alternative processes including, but not limited to, T-junction and direct injection, may be used to achieve the same nanoprecipitation.
- Vaccines of the present disclosure are typically formulated in lipid nanoparticle.
- the lipid nanoparticle comprises at least one ionizable amino lipid, at least one non-cationic lipid, at least one sterol, and/or at least one polyethylene glycol (PEG)-modified lipid.
- the lipid nanoparticle comprises 20-60 mol% ionizable amino lipid.
- the lipid nanoparticle may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% ionizable amino lipid.
- the lipid nanoparticle comprises 20 mol%, 30 mol%, 40 mol%, 50, or 60 mol% ionizable amino lipid. In some embodiments, the lipid nanoparticle comprises 5-25 mol% non-cationic lipid.
- the lipid nanoparticle may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% non-cationic lipid. In some embodiments, the lipid nanoparticle comprises 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% non-cationic lipid.
- the lipid nanoparticle comprises 25-55 mol% sterol.
- the lipid nanoparticle may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% sterol.
- the lipid nanoparticle comprises 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% sterol.
- the lipid nanoparticle comprises 0.5-15 mol% PEG-modified lipid.
- the lipid nanoparticle may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol%.
- the lipid nanoparticle comprises 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% PEG-modified lipid.
- the lipid nanoparticle comprises 20-60 mol% ionizable amino lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-modified lipid.
- the lipid nanoparticle comprises 40-50 mol% ionizable amino lipid, 5-15 mol% neutral lipid, 20-40 mol% cholesterol, and 0.5-3 mol% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises 45-50 mol% ionizable amino lipid, 9-13 mol% neutral lipid, 35-45 mol% cholesterol, and 2-3 mol% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises 48 mol% ionizable amino lipid, 11 mol% neutral lipid, 68.5 mol% cholesterol, and 2.5 mol% PEG-modified lipid.
- an ionizable amino lipid of the disclosure comprises a compound of Formula (I): or a salt or isomer thereof, wherein: R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -O(CH 2 ) n
- a subset of compounds of Formula (I) includes those in which when R4 is -(CH2)nQ, -(CH2)nCHQR, –CHQR, or -CQ(R)2, then (i) Q is not -N(R)2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.
- another subset of compounds of Formula (I) includes those in which R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R 4 is selected from the group consisting of a C 3-6 carbocycle, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S,
- another subset of compounds of Formula (I) includes those in which R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R 4 is selected from the group consisting of a C 3-6 carbocycle, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, -CQ(R) 2 , and unsubstituted C 1-6 alkyl, where Q is selected from a C 3-6 carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms selected from N, O, and
- another subset of compounds of Formula (I) includes those in which R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R 2 and R 3 are independently selected from the group consisting of H, C 1-14 alkyl, C 2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R 4 is selected from the group consisting of a C 3-6 carbocycle, -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S,
- another subset of compounds of Formula (I) includes those in which R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R2 and R3 are independently selected from the group consisting of H, C2-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4 is -(CH2)nQ or -(CH2)nCHQR, where Q is -N(R)2, and n is selected from 3, 4, and 5; each R 5 is independently selected from the group consisting of C 1-3 alkyl, C 2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M’ are independently selected from -C(
- another subset of compounds of Formula (I) includes those in which R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R 2 and R 3 are independently selected from the group consisting of C 1-14 alkyl, C 2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R 4 is selected from the group consisting of -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR, and -CQ(R)2, where Q is -N(R)2, and n is selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C
- a subset of compounds of Formula (I) includes those of Formula (IIa), (IIb), (IIc), or (IIe): or a salt or isomer thereof, wherein R 4 is as described herein.
- a subset of compounds of Formula (I) includes those of Formula (IId): (IId), or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R’, R”, and R2 through R6 are as described herein.
- each of R 2 and R 3 may be independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.
- an ionizable amino lipid of the disclosure comprises a compound having structure: In some embodiments, an ionizable amino lipid of the disclosure comprises a compound having structure: In some embodiments, a non-cationic lipid of the disclosure comprises 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero- phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoy
- a PEG modified lipid of the disclosure comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof.
- the PEG-modified lipid is DMG-PEG, PEG-c- DOMG (also referred to as PEG-DOMG), PEG-DSG and/or PEG-DPG.
- a sterol of the disclosure comprises cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and mixtures thereof.
- a LNP of the disclosure comprises an ionizable amino lipid of Compound 1, wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG lipid is DMG-PEG (e.g., PEG2000-DMG).
- the lipid nanoparticle comprises 45 – 55 mole percent (mol%) ionizable amino lipid (e.g., Compound 1).
- lipid nanoparticle may comprise 45-47, 45-48, 45-49, 45-50, 45-52, 46-48, 46-49, 46-50, 46-52, 46-55, 47-48, 47-49, 47-50, 47-52, 47- 55, 48-50, 48-52, 48-55, 49-50, 49-52, 49-55, or 50-55 mol% ionizable amino lipid (e.g., Compound 1).
- lipid nanoparticle may comprise 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% ionizable amino lipid.
- the lipid nanoparticle comprises 5 – 15 mol% non-cationic (neutral) lipid (e.g., DSPC).
- the lipid nanoparticle may comprise 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, 6-15, 7-8, 7-9, 7- 10, 7-11, 7-12, 7-13, 7-14, 7-15, 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 10-11, 10-12, 10-13, 10-14, 10-15, 11-12, 11-13, 11-14, 11-15, 12-13, 12-14, 13-14, 13-15, or 14-15 mol% non-cationic (neutral) lipid (e.g., DSPC).
- the lipid nanoparticle may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% DSPC.
- the lipid nanoparticle comprises 35 – 40 mol% sterol (e.g., cholesterol).
- the lipid nanoparticle may comprise 35-36, 35-37, 35-38, 35-39, 35- 40, 36-37, 36-38, 36-39, 36-40, 37-38, 37-39, 37-40, 38-39, 38-40, or 39-40 mol% cholesterol.
- the lipid nanoparticle may comprise 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, or 40 mol% cholesterol.
- the lipid nanoparticle comprises 1 – 3 mol% DMG-PEG.
- the lipid nanoparticle may comprise 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3.
- mol% DMG-PEG mol% DMG-PEG.
- the lipid nanoparticle may comprise 1, 1.5, 2, 2.5, or 3 mol% DMG-PEG.
- the lipid nanoparticle comprises 50 mol% ionizable amino lipid, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.
- the lipid nanoparticle comprises 48 mol% ionizable amino lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% PEG2000-DMG.
- an LNP of the disclosure comprises an N:P ratio of from about 2:1 to about 30:1.
- an LNP of the disclosure comprises an N:P ratio of about 6:1.
- an LNP of the disclosure comprises an N:P ratio of about 3:1.
- an LNP of the disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of from about 10:1 to about 100:1.
- an LNP of the disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of about 20:1. In some embodiments, an LNP of the disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of about 10:1. In some embodiments, an LNP of the disclosure has a mean diameter from about 50 nm to about 150 nm. In some embodiments, an LNP of the disclosure has a mean diameter from about 70 nm to about 120 nm.
- compositions may include RNA or multiple RNAs encoding two or more antigens of the same or different species; that is, the compositions may be multivalent compositions (e.g., vaccines).
- the composition includes an RNA or multiple RNAs encoding two or more orthopoxvirus antigens.
- the RNA may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more orthopoxvirus antigens.
- two or more different mRNA encoding antigens may be formulated in the same lipid nanoparticle.
- compositions e.g., pharmaceutical compositions
- methods, kits and reagents for prevention or treatment of orthopoxviruses in humans and other mammals for example.
- the compositions provided herein can be used as therapeutic or prophylactic agents. They may be used in medicine to prevent and/or treat an orthopoxvirus infection.
- the orthopoxvirus vaccine containing RNA as described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the RNA polynucleotides are translated in vivo to produce an antigenic polypeptide (antigen).
- a composition e.g., comprising RNA
- An “effective amount” of a composition is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the RNA (e.g., length, nucleotide composition, and/or extent of modified nucleosides), other components of the vaccine, and other determinants, such as age, body weight, height, sex and general health of the subject.
- an effective amount of a composition provides an induced or boosted immune response as a function of antigen production in the cells of the subject.
- an effective amount is the amount necessary to prevent infection or reduce the severity of an orthopoxvirus infection in the subject based on a single dose of the vaccine or single dose of the vaccine with a booster dose.
- an effective amount of the composition containing RNA polynucleotides having at least one chemical modification are more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or a peptide antigen.
- Increased antigen production may be demonstrated by increased cell transfection (the percentage of cells transfected with the RNA vaccine), increased protein translation and/or expression from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by increased duration of protein translation from a modified polynucleotide), or altered antigen specific immune response of the host cell.
- pharmaceutical composition refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.
- a "pharmaceutically acceptable carrier,” after administered to or upon a subject, does not cause undesirable physiological effects.
- the carrier in the pharmaceutical composition must be "acceptable” also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it.
- One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent.
- a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form.
- examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.
- compositions comprising polynucleotides and their encoded polypeptides in accordance with the present disclosure may be used for treatment or prevention of an orthopoxvirus infection.
- a composition may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase or during active infection after onset of symptoms.
- the amount of RNA provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis.
- a vaccine may be administered to a subject to induce an antigen specific immune response, as a singular vaccine (i.e., where both mRNAs encoding antigens are included in the same formulation) or as separate vaccines (i.e., the mRNA encoding a first antigen and the mRNA encoding a second antigen are administered separately).
- the vaccine is administered as a separate vaccine, the two mRNAs may be administered to the subject at the same time (i.e., within an hour of one another) or at different times (i.e., separated by more than an hour, 12 hours, 24 hours, 2 days, 7 days, 2 weeks).
- the two mRNAs may be administered to the subject at the same site or a different site (i.e., as an injection in separate arms).
- the vaccine may be the only vaccine comprising a nucleic acid encoding an orthopoxvirus antigen that a subject receives.
- the vaccine may be administered in various combinations, as a prime and/or boost dose.
- the vaccine may be administered to seropositive or seronegative subjects.
- a subject may be na ⁇ ve and not have antibodies that react with a virus having an antigen, wherein the antigen is the viral antigen or fragment thereof encoded by the mRNA of the vaccine. Such a subject is said to be seronegative with respect to that vaccine.
- the subject may have preexisting antibodies to viral antigen encoded by the mRNA of the vaccine because they have previously had an infection with virus carrying the antigen or may have previously been administered a dose of a vaccine (e.g., an mRNA vaccine) that induces antibodies against the antigen.
- a vaccine e.g., an mRNA vaccine
- Such a subject is said to be seropositive with respect to that vaccine.
- the subject may have been previously exposed to a virus but not to a specific variant or strain of the virus or a specific vaccine associated with that variant or strain.
- Such a subject is considered to be seronegative with respect to the specific variant or strain.
- compositions e.g., mRNA vaccines
- compositions e.g., mRNA vaccines
- compositions can be administered to seropositive or seronegative subjects in some embodiments.
- a seronegative subject may be na ⁇ ve and not have antibodies that react with the specific virus which the subject is being immunized against.
- a seropositive subject may have preexisting antibodies to the specific virus because they have previously had an infection with that virus, variant or strain or may have previously been administered a dose of a vaccine (e.g., an mRNA vaccine) that induces antibodies against that virus, variant or strain.
- a composition includes mRNA encoding at least one (e.g., one, two, or more) orthopoxvirus antigens, such as monkeypox virus antigens from different monkeypox mutant strains (also referred to herein as variants).
- the mRNA vaccine comprises multiple mRNAs encoding monkeypox antigens from different variants in a single lipid nanoparticle.
- a composition may be administered with other prophylactic or therapeutic compounds.
- a prophylactic or therapeutic compound may be an adjuvant or a booster.
- the term “booster” or “booster vaccine” refers to an extra administration of the prophylactic vaccine composition.
- the booster vaccine comprises at least one mRNA polynucleotide having an ORF encoding the first, second or third an orthopoxvirus antigenic polypeptides.
- the booster vaccine comprises at least one mRNA polynucleotide having an ORF encoding each of the first, second and third an orthopoxvirus antigenic polypeptides. In some embodiments, the booster vaccine comprises at least one mRNA polynucleotide having an ORF encoding a variant of the first, second or third an orthopoxvirus antigenic polypeptides.
- a booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition.
- the time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months.
- the time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or 6 months.
- the booster vaccine is administered between three weeks and one year after the vaccine.
- a composition may be administered intramuscularly, intranasally or intradermally, similarly to the administration of inactivated vaccines known in the art.
- a composition may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need.
- the RNA vaccines may be utilized to treat and/or prevent a variety of infectious disease.
- RNA vaccines have superior properties in that they produce much larger antibody titers, better neutralizing immunity, produce more durable immune responses, and/or produce responses earlier than commercially available vaccines.
- pharmaceutical compositions including RNA and/or complexes optionally in combination with one or more pharmaceutically acceptable excipients.
- the RNA may be formulated or administered alone or in conjunction with one or more other components.
- an immunizing composition may comprise other components including, but not limited to, adjuvants.
- an immunizing composition does not include an adjuvant (they are adjuvant free).
- An RNA may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients.
- vaccine compositions comprise at least one additional active substance, such as, for example, a therapeutically-active substance, a prophylactically-active substance, or a combination of both.
- Vaccine compositions may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents, such as vaccine compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).
- an immunizing composition is administered to humans, human patients or subjects.
- the phrase “active ingredient” generally refers to the RNA vaccines or the polynucleotides contained therein, for example, RNA polynucleotides (e.g., mRNA polynucleotides) encoding antigens.
- Formulations of the vaccine compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., mRNA polynucleotide) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
- compositions in accordance with the disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
- the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
- an RNA is formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation); (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein (antigen) in vivo.
- excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the RNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.
- immunizing compositions e.g., RNA vaccines
- Immunizing compositions can be used as therapeutic or prophylactic agents.
- immunizing compositions are used to provide prophylactic protection from orthopoxvirus infections.
- immunizing compositions are used to treat orthopoxvirus infections.
- immunizing compositions are used to reduce the severity of an orthopoxvirus infection in a subject.
- immunizing compositions are used in the priming of immune effector cells, for example, to activate peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re- infused) into a subject.
- PBMCs peripheral blood mononuclear cells
- a subject may be any mammal, including non-human primate and human subjects.
- a subject is a human subject.
- the subject is 60 years of age or older (e.g., 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 years of age or older).
- the subject is under 18 years of age (e.g., under 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years of age).
- an immunizing composition e.g., RNA a vaccine
- a subject e.g., a mammalian subject, such as a human subject
- the RNA encoding the orthopoxvirus antigen is expressed and translated in vivo to produce the antigen, which then stimulates an immune response in the subject.
- Prophylactic protection from an orthopoxvirus can be achieved following administration of an immunizing composition (e.g., an RNA vaccine) of the present disclosure.
- Immunizing compositions can be administered once, twice, three times, four times or more but it is likely sufficient to administer the vaccine once (optionally followed by a single booster).
- a method of eliciting an immune response in a subject against an orthopoxvirus antigen is provided in aspects of the present disclosure.
- a method involves administering to the subject an immunizing composition comprising a mRNA having an open reading frame encoding an orthopoxvirus antigen, thereby inducing in the subject an immune response specific to the orthopoxvirus antigen, wherein anti-antigen antibody titer in the subject is increased following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the antigen.
- An “anti-antigen antibody” is a serum antibody the binds specifically to the antigen.
- a prophylactically effective dose is an effective dose that prevents infection with the virus at a clinically acceptable level.
- the effective dose is a dose listed in a package insert for the vaccine.
- a traditional vaccine refers to a vaccine other than the mRNA vaccines of the present disclosure.
- a traditional vaccine includes, but is not limited, to live microorganism vaccines, killed microorganism vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, virus like particle (VLP) vaccines, etc.
- a traditional vaccine is a vaccine that has achieved regulatory approval and/or is registered by a national drug regulatory body, for example the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA).
- FDA Food and Drug Administration
- EMA European Medicines Agency
- the anti-antigen antibody titer in the subject is increased 1 log to 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the orthopoxvirus or an unvaccinated subject. In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log, 2 log, 3 log, 4 log, 5 log, or 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the orthopoxvirus or an unvaccinated subject.
- a method of eliciting an immune response in a subject against an orthopoxvirus involves administering to the subject an immunizing composition (e.g., an RNA vaccine) comprising a RNA polynucleotide comprising an open reading frame encoding an orthopoxvirus antigen, thereby inducing in the subject an immune response specific to the orthopoxvirus, wherein the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine against the orthopoxvirus at 2 times to 100 times the dosage level relative to the immunizing composition.
- an immunizing composition e.g., an RNA vaccine
- a RNA polynucleotide comprising an open reading frame encoding an orthopoxvirus antigen
- the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at twice the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at three times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 4 times, 5 times, 10 times, 50 times, or 100 times the dosage level relative to an immunizing composition of the present disclosure.
- the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 10 times to 1000 times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 100 times to 1000 times the dosage level relative to an immunizing composition of the present disclosure. In other embodiments, the immune response is assessed by determining [protein] antibody titer in the subject. In other embodiments, the ability to promote a robust T cell response(s) is measured using art recognized techniques.
- the disclosure provide methods of eliciting an immune response in a subject against an orthopoxvirus by administering to the subject an immunizing composition (e.g., an RNA vaccine) comprising an RNA having an open reading frame encoding an orthopoxvirus antigen, thereby inducing in the subject an immune response specific to the orthopoxvirus antigen, wherein the immune response in the subject is induced 2 days to 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the orthopoxvirus.
- an immunizing composition e.g., an RNA vaccine
- the immune response in the subject is induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine at 2 times to 100 times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is induced 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.
- RNA vaccines may be administered by any route that results in a therapeutically effective outcome. These include, but are not limited, to intradermal, intramuscular, intranasal, and/or subcutaneous administration.
- the present disclosure provides methods comprising administering RNA vaccines to a subject in need thereof.
- RNA is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the RNA may be decided by the attending physician within the scope of sound medical judgment.
- the specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
- the effective amount of the RNA, as provided herein may be as low as 25 ⁇ g (total mRNA), administered for example as a single dose or as two 12.5 ⁇ g doses.
- a “dose” as used herein, represents the sum total of RNA in the composition (e.g., including all of the NA antigens and/or HA antigens in the formulation).
- the effective amount is a total dose of 25 ⁇ g-300 ⁇ g, 50 ⁇ g-300 ⁇ g, 100 ⁇ g -300 ⁇ g, 150 ⁇ g -300 ⁇ g, 200 ⁇ g -300 ⁇ g, 250 ⁇ g - 300 ⁇ g, 150 ⁇ g -200 ⁇ g, 150 ⁇ g -250 ⁇ g, 150 ⁇ g -300 ⁇ g, 200 ⁇ g -250 ⁇ g, or 250 ⁇ g -300 ⁇ g.
- the effective amount may be a total dose of 25 ⁇ g, 50 ⁇ g, 55 ⁇ g, 60 ⁇ g, 65 ⁇ g, 70 ⁇ g, 75 ⁇ g, 80 ⁇ g, 85 ⁇ g, 90 ⁇ g, 95 ⁇ g, 100 ⁇ g, 110 ⁇ g, 120 ⁇ g, 130 ⁇ g, 140 ⁇ g, 150 ⁇ g, 160 ⁇ g, 170 ⁇ g, 180 ⁇ g, 190 ⁇ g, 200 ⁇ g, 210 ⁇ g, 220 ⁇ g, 230 ⁇ g, 240 ⁇ g, 250 ⁇ g, 260 ⁇ g, 270 ⁇ g, 280 ⁇ g, 290 ⁇ g, or 300 ⁇ g.
- the effective amount is a total dose of 25 ⁇ g. In some embodiments, the effective amount is a total dose of 30 ⁇ g. In some embodiments, the effective amount is a total dose of 50 ⁇ g. In some embodiments, the effective amount is a total dose of 66 ⁇ g. In some embodiments, the effective amount is a total dose of 67 ⁇ g. In some embodiments, the effective amount is a total dose of 68 ⁇ g. In some embodiments, the effective amount is a total dose of 132 ⁇ g. In some embodiments, the effective amount is a total dose of 133 ⁇ g. In some embodiments, the effective amount is a total dose of 134 ⁇ g.
- the effective amount is a total dose of 266 ⁇ g. In some embodiments, the effective amount is a total dose of 267 ⁇ g. In some embodiments, the effective amount is a total dose of 268 ⁇ g. In some embodiments, the effective amount is a total dose of 100 ⁇ g. In some embodiments, the effective amount is a total dose of 200 ⁇ g. In some embodiments, the effective amount is a total dose of 300 ⁇ g.
- RNA described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).
- Vaccine Efficacy Some aspects of the present disclosure provide formulations of the immunizing compositions (e.g., RNA vaccines), wherein the RNA is formulated in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to an orthopoxvirus antigen). “An effective amount” is a dose of the RNA effective to produce an antigen-specific immune response.
- an immune response to a vaccine or LNP of the present disclosure is the development in a subject of a humoral and/or a cellular immune response to a (one or more) orthopoxvirus protein(s) present in the vaccine.
- a “humoral” immune response refers to an immune response mediated by antibody molecules, including, e.g., secretory (IgA) or IgG molecules, while a “cellular” immune response is one mediated by T-lymphocytes (e.g., CD4+ helper and/or CD8+ T cells (e.g., CTLs) and/or other white blood cells.
- CTLs cytolytic T-cells
- MHC major histocompatibility complex
- helper T-cells act to help stimulate the function and focus the activity nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface.
- a cellular immune response also leads to the production of cytokines, chemokines, and other such molecules produced by activated T-cells and/or other white blood cells including those derived from CD4+ and CD8+ T-cells.
- the antigen-specific immune response is characterized by measuring an anti-orthopoxvirus antigen antibody titer produced in a subject administered an immunizing composition as provided herein.
- An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen or epitope of an antigen.
- Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result.
- Enzyme-linked immunosorbent assay is a common assay for determining antibody titers, for example.
- an antibody titer is used to assess whether a subject has had an infection or to determine whether immunizations are required.
- an antibody titer is used to determine the strength of an autoimmune response, to determine whether a booster immunization is needed, to determine whether a previous vaccine was effective, and to identify any recent or prior infections.
- an antibody titer may be used to determine the strength of an immune response induced in a subject by an immunizing composition (e.g., RNA vaccine).
- an immunizing composition e.g., RNA vaccine
- an anti-orthopoxvirus antigen antibody titer produced in a subject is increased by at least 1 log relative to a control.
- anti-orthopoxvirus antigen antibody titer produced in a subject may be increased by at least 1.5, at least 2, at least 2.5, or at least 3 log relative to a control.
- the anti-orthopoxvirus antigen antibody titer produced in the subject is increased by 1, 1.5, 2, 2.5 or 3 log relative to a control.
- the anti-orthopoxvirus antigen antibody titer produced in the subject is increased by 1-3 log relative to a control.
- the anti-orthopoxvirus virus antigen antibody titer produced in a subject may be increased by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to a control.
- the anti-orthopoxvirus antigen antibody titer produced in a subject is increased at least 2 times relative to a control.
- the anti-orthopoxvirus antigen antibody titer produced in a subject may be increased at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times relative to a control.
- the anti-orthopoxvirus antigen antibody titer produced in the subject is increased 2, 3, 4, 5, 6, 7, 8, 9, or 10 times relative to a control. In some embodiments, the anti-orthopoxvirus antigen antibody titer produced in a subject is increased 2- 10 times relative to a control.
- the anti-orthopoxvirus antigen antibody titer produced in a subject may be increased 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times relative to a control.
- an antigen-specific immune response is measured as a ratio of geometric mean titer (GMT), referred to as a geometric mean ratio (GMR), of serum neutralizing antibody titers to an orthopoxvirus.
- GTT geometric mean titer
- a geometric mean titer (GMT) is the average antibody titer for a group of subjects calculated by multiplying all values and taking the nth root of the number, where n is the number of subjects with available data.
- a control in some embodiments, is an anti-orthopoxvirus antigen antibody titer produced in a subject who has not been administered an immunizing composition (e.g., RNA vaccine).
- a control is an anti-orthopoxvirus antigen antibody titer produced in a subject administered a recombinant or purified protein vaccine.
- Recombinant protein vaccines typically include protein antigens that either have been produced in a heterologous expression system (e.g., bacteria or yeast) or purified from large amounts of the pathogenic organism.
- the ability of an immunizing composition e.g., RNA vaccine
- an immunizing composition may be administered to a murine model and the murine model assayed for induction of neutralizing antibody titers. Viral challenge studies may also be used to assess the efficacy of a vaccine of the present disclosure.
- an immunizing composition may be administered to a murine model, the murine model challenged with virus, and the murine model assayed for survival and/or immune response (e.g., neutralizing antibody response, T cell response (e.g., cytokine response)).
- a “standard of care,” as provided herein, refers to a medical or psychological treatment guideline and can be general or specific. “Standard of care” specifies appropriate treatment based on scientific evidence and collaboration between medical professionals involved in the treatment of a given condition. It is the diagnostic and treatment process that a physician/ clinician should follow for a certain type of patient, illness or clinical circumstance.
- a “standard of care dose,” as provided herein, refers to the dose of a recombinant or purified protein vaccine, or a live attenuated or inactivated vaccine, or a VLP vaccine, that a physician/clinician or other medical professional would administer to a subject to treat or prevent an orthopoxvirus infection or a related condition, while following the standard of care guideline for treating or preventing an orthopoxvirus infection or a related condition.
- the anti-orthopoxvirus antigen antibody titer produced in a subject administered an effective amount of an immunizing composition is equivalent to an anti- orthopoxvirus antigen antibody titer produced in a control subject administered a standard of care dose of a recombinant or purified protein vaccine, or a live attenuated or inactivated vaccine, or a VLP vaccine.
- Vaccine efficacy may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis.2010 Jun 1;201(11):1607-10). For example, vaccine efficacy may be measured by double-blind, randomized, clinical controlled trials.
- AR disease attack rate
- RR relative risk
- vaccine effectiveness may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis.2010 Jun 1;201(11):1607-10).
- Vaccine effectiveness is an assessment of how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population.
- Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non-vaccine-related factors that influence the ‘real-world’ outcomes of hospitalizations, ambulatory visits, or costs.
- a retrospective case control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared.
- efficacy of the immunizing composition is at least 60% relative to unvaccinated control subjects.
- efficacy of the immunizing composition may be at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 98%, or 100% relative to unvaccinated control subjects.
- Sterilizing Immunity refers to a unique immune status that prevents effective pathogen infection into the host.
- the effective amount of an immunizing composition of the present disclosure is sufficient to provide sterilizing immunity in the subject for at least 1 year.
- the effective amount of an immunizing composition of the present disclosure is sufficient to provide sterilizing immunity in the subject for at least 2 years, at least 3 years, at least 4 years, or at least 5 years.
- the effective amount of an immunizing composition of the present disclosure is sufficient to provide sterilizing immunity in the subject at an at least 5-fold lower dose relative to control.
- the effective amount may be sufficient to provide sterilizing immunity in the subject at an at least 10-fold lower, 15-fold, or 20-fold lower dose relative to a control.
- Detectable Antigen In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to produce detectable levels orthopoxvirus antigen as measured in serum of the subject at 1-72 hours post administration. Titer.
- an antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen (e.g., an anti-orthopoxvirus antigen). Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example.
- the effective amount of an immunizing composition of the present disclosure is sufficient to produce a 1,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the orthopoxvirus antigen as measured in serum of the subject at 1- 72 hours post administration.
- the effective amount is sufficient to produce a 1,000-5,000 neutralizing antibody titer produced by neutralizing antibody against the orthopoxvirus antigen as measured in serum of the subject at 1-72 hours post administration. In some embodiments, the effective amount is sufficient to produce a 5,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the orthopoxvirus antigen as measured in serum of the subject at 1-72 hours post administration. In some embodiments, the neutralizing antibody titer is at least 100 NT 50 . For example, the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NT50. In some embodiments, the neutralizing antibody titer is at least 10,000 NT50.
- the neutralizing antibody titer is at least 100 neutralizing units per milliliter (NU/mL).
- the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NU/mL.
- the neutralizing antibody titer is at least 10,000 NU/mL.
- an anti-orthopoxvirus antigen antibody titer produced in the subject is increased by at least 1 log relative to a control.
- an anti-orthopoxvirus antigen antibody titer produced in the subject may be increased by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 log relative to a control.
- an anti-orthopoxvirus antigen antibody titer produced in the subject is increased at least 2 times relative to a control.
- an anti-orthopoxvirus antigen antibody titer produced in the subject is increased by at least 3, 4, 5, 6, 7, 8, 9 or 10 times relative to a control.
- a geometric mean which is the nth root of the product of n numbers, is generally used to describe proportional growth. Geometric mean, in some embodiments, is used to characterize antibody titer produced in a subject.
- a control may be, for example, an unvaccinated subject, or a subject administered a live attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit vaccine.
- a monkeypox mRNA-lipid nanoparticle vaccine targeting virus binding, entry, and transmission drives protection against lethal orthopoxviral challenge
- bioRxiv 2022.12.17.520886 an mRNA-lipid nanoparticle vaccine encoding a set of four highly conserved monkeypox virus surface proteins involved in virus attachment, entry and transmission were demonstrated to induce MPXV-specific immunity and heterologous protection against a lethal vaccinia virus (VACV) challenge.
- VACV lethal vaccinia virus
- the effect of the mRNA vaccine was compared to the current MPXV vaccine (Modified Vaccinia Virus Ankara (MVA)), and produced superior neutralizing and cellular spread-inhibitory activities against MPXV and VACV as well as greater Fc-effector Th1-biased humoral immunity to the four MPXV antigens and the four VACV homologs.
- Combinations of two, three or four MPXV antigen expressing mRNAs protected against disease-related weight loss and death.
- Multivalent MPXV mRNAs also resulted in superior cross-protection compared to MVA.
- the cross-protective response was associated with a combination of neutralizing and non-neutralizing antibody functions.
- an MPXV mRNA vaccine can induce robust neutralizing and functional cross-reactive antibodies able to confer comparable, if not superior, protection against a lethal challenge of an orthologous orthopoxvirus compared to MVA.
- any of the mRNA sequences described herein may include a 5’ UTR and/or a 3’ UTR.
- the UTR sequences may be selected from any of the UTR sequences disclosed herein, or other known UTR sequences may be used. It should also be understood that any of the mRNAs described herein may further comprise a poly(A) tail and/or cap (e.g., 7mG(5’)ppp(5’)NlmpNp).
- RNAs and encoded antigen sequences described herein include a signal peptide and/or a peptide tag (e.g., C-terminal His tag), it should be understood that the indicated signal peptide and/or peptide tag may be substituted for a different signal peptide and/or peptide tag, or the signal peptide and/or peptide tag may be omitted.
- a signal peptide and/or a peptide tag e.g., C-terminal His tag
Abstract
The disclosure provides mRNA vaccines for orthopoxviruses, such as smallpox and monkeypox, as well as methods of using the vaccines.
Description
ORTHOPOXVIRUS VACCINES RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 63/345,390 filed May 24, 2022 and U.S. provisional application number 63/395,567 filed August 05, 2022, each of which is incorporated by reference herein in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (M137870248WO00-SEQ-HCL.xml; Size: 128,336 bytes; and Date of Creation: May 16, 2023) is herein incorporated by reference in its entirety. BACKGROUND Poxvirus (a member of the Poxviridae) is a double-stranded DNA virus that infects both humans and animals. Poxviruses are divided into two subfamilies based on host range: the Acrididae (Chordopoxviridae) family includes four genera, orthopoxvirus genus (Orthopoxvirus), Parapoxvirus (Parapoxvirus), molluscum pox virus (Molluscipoxvirus) and Yatapoxvirus, that are known to infect humans. The Orthopoxvirus genera includes a number of genetically related and morphologically identical viruses, including variola virus (VARV), camel pox virus (CMLV), vaccinia virus (CPXV), apoplexy virus (ECTV), horse pox virus (HPXV), and monkeypox virus (MPXV), rabbit pox virus (RPXV), raccoon pox virus, skunk pox virus, gerbil pox virus, Uasin Gishu disease virus, vaccinia virus (VACV), smallpox virus (VARV), and volepox virus (VPV). At least four viruses are known to infect humans: VARV, VACV, MPXV, and CPXV. Currently, “live” vaccination is the only proven preventive measure against smallpox. Due to a comprehensive vaccination program, smallpox was eradicated in 1977, and routine vaccination of the public against smallpox ended. Unfortunately, outbreaks of orthopoxviruses persist; for example, several clusters of monkeypox have been reported in May 2022. While monkeypox transmits poorly from person to person and has a lower rate of mortality (4-15%) compared to smallpox (30%), in contrast to smallpox, monkeypox cannot be eradicated. The virus has an unknown animal reservoir, and the existence of more virulent strains is possible. The continuing health problems associated with orthopoxviruses, such as monkeypox and smallpox, are of concern internationally, reinforcing the importance of developing effective and safe vaccine candidates against these viruses.
SUMMARY Provided herein, in some embodiments, are compositions (e.g., vaccines) that comprise one or more messenger ribonucleic acid (mRNA) molecules that encode(s) highly immunogenic antigen(s) capable of eliciting potent neutralizing antibody responses against orthopoxvirus (e.g., smallpox, monkeypox) antigens. The mRNA molecules described herein are used to express key antigenic components of the virus (e.g., protein ectodomains of A35L and B6R derived from extracellular virus (EV), M1R and A29L derived from mature virus (MV)) that are efficient at inducing protective immunity when used individually or in combination as an immunogenic composition or vaccine to protect people from infection by the natural virus and/or to reduce symptoms if infected. Thus, some aspects of the present disclosure provide compositions comprising an mRNA encoding a functional domain of an orthopoxvirus (e.g., smallpox, monkeypox) capable of inducing an immune response, such as a neutralizing antibody response, to an orthopoxvirus (e.g., smallpox, monkeypox). In some embodiments, the mRNA is formulated in a lipid nanoparticle. Some aspects of the disclosure provide a composition, comprising a first messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame (ORF) encoding a first orthopoxvirus protein and a lipid nanoparticle. In some embodiments, the composition further comprises a second mRNA polynucleotide comprising an ORF encoding a second orthopoxvirus protein. In some embodiments, the composition further comprises a third mRNA polynucleotide comprising an ORF encoding a third orthopoxvirus protein. In some embodiments, the composition further comprises a fourth mRNA polynucleotide comprising an ORF encoding a fourth orthopoxvirus protein. In some embodiments, the first orthopoxvirus protein comprises a mature virus (MV) orthopoxvirus protein or an extracellular enveloped virus (EV) orthopoxvirus protein. In some embodiments, the second orthopoxvirus protein comprises a MV orthopoxvirus protein or an EV orthopoxvirus protein. In some embodiments, the third orthopoxvirus protein comprises an MV orthopoxvirus protein or an EV orthopoxvirus protein. In some embodiments, the fourth orthopoxvirus protein comprises an MV orthopoxvirus protein or an EV orthopoxvirus protein. In some embodiments, the MV orthopoxvirus protein is A29L or M1R. In some embodiments, the EV orthopoxvirus protein is B6R or A35R. In some embodiments, the first orthopoxvirus protein comprises A27L, the second orthopoxvirus protein comprises M1R, the third orthopoxvirus protein comprises B6R, and the fourth orthopoxvirus protein comprises A35R.
The disclosure, in some aspects, provides a composition comprising a first messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame (ORF) encoding a first orthopoxvirus protein, and a lipid nanoparticle, wherein the first orthopoxvirus protein is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 7-34 or an immunogenic fragment thereof. In some embodiments, the composition further comprises a second mRNA polynucleotide comprising an ORF encoding a second orthopoxvirus protein, wherein the second orthopoxvirus protein is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 7-34 or an immunogenic fragment thereof. In some embodiments, the composition further comprises a third mRNA polynucleotide comprising an ORF encoding a third orthopoxvirus protein, wherein the third orthopoxvirus protein is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 7-34 or an immunogenic fragment thereof. In some embodiments, the composition further comprises a fourth mRNA polynucleotide comprising an ORF encoding a fourth orthopoxvirus protein, wherein the fourth orthopoxvirus protein is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 7-34 or an immunogenic fragment thereof. In some embodiments, the orthopoxvirus protein is at least 90%, at least 95%, at least 97%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 7-34. In some embodiments, the orthopoxvirus protein is identical to any one of the amino acid sequences of SEQ ID NOs: 7-34. In some embodiments, the ORF comprises a sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical to any one of SEQ ID NOs: 35-62. In some embodiments, the ORF comprises a sequence that identical to any one of SEQ ID NOs: 35-62. In some embodiments, the first, second, third, and/or fourth mRNA comprises a chemical modification. In some embodiments, the first, second, third, and/or fourth mRNA is fully modified. In some embodiments, the chemical modification is 1-methylpseudouridine. In some embodiments, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable amino lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises 1-5 mol% PEG-modified lipid; 10-20 mol% non- cationic lipid; 35-45 mol% sterol; and 40-50 mol% ionizable cationic lipid. In some embodiments, the PEG-modified lipid is 1,2 dimyristoyl-sn-glycerol, methoxypolyethyleneglycol (PEG2000 DMG), the non-cationic lipid is 1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC), the sterol is cholesterol; and the ionizable cationic lipid has the structure of Compound 1:
(Compound 1). In some aspects, the disclosure provides a method for vaccinating a subject, comprising administering to the subject any one of the compositions described herein. In some embodiments, the method prevents an orthopoxvirus infection in the subject. In some embodiments, the method reduces the severity of an orthopoxvirus infection in the subject. In some embodiments, the subject is seronegative for an orthopoxvirus. In some embodiments, the subject is seropositive for an orthopoxvirus. The disclosure, in some aspects, provides multivalent RNA composition, comprising at least two messenger ribonucleic acid (mRNA) polynucleotides, each comprising an open reading frame (ORF) encoding a monkeypox antigen and a lipid nanoparticle. In some embodiments, the monkeypox antigen comprises a mature virus (MV) orthopoxvirus protein and/or an extracellular enveloped virus (EV) orthopoxvirus protein. The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the detailed description of several embodiments, and also from the appended claims. DETAILED DESCRIPTION Orthopoxvirus is a genus of viruses in the family Poxviridae and subfamily Chordopoxvirinae. Vertebrates, including mammals and humans, and arthropods serve as natural hosts. There are at least 12 species in this genus, four of which can infect humans (smallpox virus, monkeypox virus, vaccina virus, and cowpox virus). Smallpox spread between subjects is typically respiratory, although contact with infectious skin lesions or scabs has been reported. As noted above, smallpox was eradicated in the late 1970s. Infections with wild vaccina- like viruses have been reported among cattle and buffalo herders in India and in dairy workers in Brazil and Colombia. By touching infected bovine, most humans acquire localized, cutaneous infections; however, those who are immunocompromised are at increased risk of developing systemic illness. Monkeypox is endemic to tropical forested regions of West and Central Africa (e.g., the Congo Basin) and Nigeria is in the midst of an ongoing large outbreak that started in 2017. The viral infections can cause fever, malaise, head and body aches, vomiting, and a vesiculopustular rash, and in severe cases, death. In earlier versions of vaccine candidates, the identification of target antigens was challenging. Poxviruses encode hundreds of proteins and have two infectious forms:
intracellular mature virus (MV) and extracellular enveloped virus (EV). MV is an enveloped virus with many surface proteins required for infectivity (e.g., LIR, A27L, A17L, D8L and H3L), while EV has an additional membrane surrounding the MV particle and another set of unique membrane proteins (e.g., A33R and B5R). Both EV and MV are important in viral acquisition and spread. The present disclosure therefore provides, in some embodiments, vaccines that comprise RNA (e.g., mRNA) polynucleotides encoding at least one of the following antigens: A35R (from EV; ortholog of A33), B6R (from EV; ortholog of B5), M1R (from MV; ortholog of L1R), and A29L (from MV; ortholog of A27L). In some embodiments, the vaccine comprises RNA (e.g., mRNA) polynucleotides encoding at least two of the following antigens: A35R, B6R, M1R, and A29L. In some embodiments, the vaccine comprises RNA (e.g., mRNA) polynucleotides encoding at least three of the following antigens: A35R, B6R, M1R, and A29L. In some embodiments, the vaccine comprises RNA (e.g., mRNA) polynucleotides encoding the following antigens: A35R, B6R, M1R, and A29L. In some embodiments, the vaccine comprises RNA (e.g., mRNA) polynucleotides encoding at least on MV antigen and at least one EV antigen. Also provided herein are methods of administering the vaccines, methods of producing the vaccines, compositions comprising the vaccines, and nucleic acids encoding the vaccines. The vaccines described herein may be used to induce a balanced immune response, comprising both cellular and humoral immunity, without many of the risks associated with DNA vaccination. Such a vaccine, optionally referred to herein as a multivalent vaccine, can be administered to seropositive or seronegative subjects. For example, a subject may be naïve and not have antibodies that react with at least one of the antigenic polypeptides of the vaccine, or may have preexisting antibodies to at least one of antigens of the vaccine because they have previously had an infection with the orthopoxvirus or may have previously been administered a dose of a vaccine (e.g., an mRNA vaccine) that induces antibodies against the orthopoxvirus. In some embodiments, a subject may have preexisting antibodies to all of the antigens of the vaccine. Antigens Antigens, as used herein, are proteins capable of inducing an immune response (e.g., causing an immune system to produce antibodies against the antigens). The vaccines of the present disclosure provide a unique advantage over traditional protein-based vaccination approaches in which protein antigens are purified or produced in vitro, e.g., recombinant protein production technologies. The vaccines of the present disclosure feature mRNA encoding the desired antigens, which when introduced into the body, i.e., administered to a mammalian subject
(for example a human) in vivo, cause the cells of the body to express the desired antigens. The vaccines of the present disclosure feature mRNA encoding the desired viral membrane (surface) antigens, e.g., glycoprotein antigens, which when introduced into the body, i.e., administered to a mammalian subject (for example a human) in vivo, cause the cells of the body to express the desired peptides in a native fold and, optionally with human glycosylation patterns. Thus, a vaccine encoding the viral surface antigen from a series of pathogenic viruses all presenting the properly folded and, optionally, glycosylated viral antigens in the same manner as if it were generated during an actual infection. Thus, mRNA vaccines thus offer the best vehicle for making vaccines to viruses one can produce short of using an attenuated virus, but without the associated risks. In order to facilitate delivery of the mRNAs of the present disclosure to the cells of the body, the mRNAs are encapsulated in lipid nanoparticles (LNPs). Upon delivery and uptake by cells of the body, the mRNAs are translated in the cytosol and protein or glycoprotein antigens are folded and processed by the host cell machinery. The protein and/or glycoprotein antigens are presented and elicit an adaptive humoral and cellular immune response. Neutralizing antibodies are directed against the expressed viral antigens and hence these viral protein antigens are considered the most relevant target antigens for vaccine development. Simply put, neutralizing antibodies are generally directed to the viral surface proteins which are responsible for binding to the cell and when blocked by a specific antibody, the virus is neutralized. Herein, use of the term “antigen” encompasses immunogenic viral surface proteins and immunogenic fragments (an immunogenic fragment that induces (or is capable of inducing) an immune response to a (at least one) orthopoxvirus), unless otherwise stated. In some embodiments, the antigen is a naturally occurring antigen (e.g., the antigenic polypeptide encodes a naturally occurring antigen). In some embodiments, at least one antigenic polypeptide is a non-naturally occurring antigen or an engineered version of the protein or glycoprotein antigen for use in a vaccine. In some embodiments, at least one of the antigenic polypeptides is a stabilized version of a naturally occurring antigen. In another embodiment, other modifications are engineered into the viral surface protein, such as deletion of cytoplasmic tails or mutations to facilitate protein processing or conformational stability. It should be understood that the term “protein” encompasses glycoproteins, proteins, peptides and fragments thereof and the term “antigen” encompasses antigenic portions of such molecules that provoke an immune response. For the viral vaccines included herein, the term “antigen” includes viral surface proteins, e.g., ectodomains, fragments of viral proteins (e.g., glycoproteins) and designed and or mutated versions of viral proteins (e.g., glycoproteins) derived from orthopoxviruses.
In some embodiments, the antigens are from the suspected multi-country outbreak in May of 2022 (first confirmed case was in Portugal). The 2022 genome also comprises clusters with 2018-19 monkeypox virus (MPXV) sequences from the UK, Singapore, and Israel. In some embodiments, the antigens are from the Zaire_I_96 reference strain. In some embodiments, the antigens are from the Zaire_79 strain. In some embodiments, the antigens are from the Zaire_I_96, Zaire_79 strain, the MPXV Singapore 2019 strain, and the MPXV Portugal 2022 strain, or any combination thereof. The mRNA vaccines of the instant invention comprise mRNAs encoding MV and/or EV antigens of an orthopoxvirus (e.g., monkeypox virus or smallpox virus). In some embodiments, the mRNA vaccine comprises mRNAs encoding an MV antigen (e.g M1R or A29L). In some embodiments, the mRNA vaccine comprises mRNA encoding an EV antigen (e.g., A35R or B6R). In some embodiments, the mRNAs encode at least one MV antigen and at least one EV antigen. In some embodiments, the mRNA encoding at least one MV antigen and the mRNA encoding at least one EV antigen are present in the vaccine at a 1:1 ratio, a 2:1 ratio, a 3:1 ratio, a 4:1 ratio, a 1:4 ratio, a 1:3 ratio, or a 1:2 ratio. In some embodiments, the antigen is an M1R protein (an ortholog of vaccinia L1R). The L1R protein is essential for vaccinia virus (VACV) replication and interacts with the entry-fusion complex (EFC). It is found on the surface of the mature virus (MV). In some embodiments, the antigen is a B6R protein (an ortholog of vaccina B5R). The protein is found on the surface of the EV. In some embodiments, the antigen is an A35R protein (an ortholog of vaccinia A33R). The protein is found on the surface of the EV. A33 is a type II integral membrane protein that forms disulfide-bonded homodimers or heteromultimers (Roper, Payne, and Moss, 1996). It coordinates the incorporation of A36 into IEV membranes and subsequently, the production of actin tails (Wolffe, Weisberg, and Moss, 2001). Deletion of A33R causes a small plaque phenotype. This phenotype is due to a number of defects, including the inability to form actin tails and a reduction in the infectiousness of EV (Chan and Ward, 2010; Roper et al., 1998). A33 is a major target for antibody neutralization of the enveloped form of the virus and is a potential candidate for a subunit vaccine against orthopoxvirus infections (Hooper, Custer, and Thompson, 2003; Hooper et al., 2004). In some embodiments, the antigen is an A29L protein (an ortholog of A27L). The A27L protein is present on the surface of the mature virus, and it mediates vaccinia virus interaction with cell surface heparan sulfate. It is required for the microtubule-dependent transport of intracellular mature virus particles, and it associates, via C-terminal leucine zipper domain (residues 85–110), with viral membrane protein A17 for anchoring to the viral membrane.
In some embodiments, the antigen is any one of the antigens provided herein. In some embodiments, the antigen is 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or identical to any one of SEQ ID NOs: 7-34. In each embodiment or aspect of the invention, it is understood that the featured vaccines include the mRNAs encapsulated within LNPs. While it is possible to encapsulate each unique mRNA (encoding its unique antigen) in its own LNP, the mRNA vaccine technology enjoys the significant technological advantage of being able to encapsulate several mRNAs in a single LNP product. Nucleic Acids The compositions of the present disclosure comprise a (at least one) messenger RNA (mRNA) having an open reading frame (ORF) encoding an orthopoxvirus (e.g., smallpox or monkeypox) antigen. In some embodiments, the ORF is 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or identical to any one of SEQ ID NOs: 35-62. In some embodiments, the mRNA further comprises a 5 ^ UTR, 3 ^ UTR, a poly(A) tail and/or a 5 ^ cap analog. In some embodiments, the first, second and/or third mRNA polynucleotides in the composition differ in length from one another by at least 100 nucleotides (e.g., 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, or more nucleotides). It should also be understood that the orthopoxvirus virus vaccine of the present disclosure may include any 5′ untranslated region (UTR) and/or any 3′ UTR. Exemplary UTR sequences include SEQ ID NOs: 1-4 and 92-93; however, other UTR sequences may be used or exchanged for any of the UTR sequences described herein. In some embodiments, a 5' UTR of the present disclosure comprises a sequence selected from SEQ ID NO: 1 (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC), SEQ ID NO: 2 (GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCC ACC), or SEQ ID NO.92 (GGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCUCGCAAC UAGCAAGCUUUUUGUUCUCGCC). In some embodiments, a 3' UTR of the present disclosure comprises a sequence selected from SEQ ID NO: 3 (UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC), SEQ ID NO: 4 (UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC
AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC) or SEQ ID NO.: 93 (UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGA GUGGGCGGC). UTRs may also be omitted from the RNA polynucleotides provided herein. Nucleic acids comprise a polymer of nucleotides (nucleotide monomers). Thus, nucleic acids are also referred to as polynucleotides. Nucleic acids may be or may include, for example, deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino- α-LNA having a 2′- amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) and/or chimeras and/or combinations thereof. Messenger RNA (mRNA) is any RNA that encodes a (at least one) protein (a naturally- occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application may recite “T”s in a representative DNA sequence but where the sequence represents mRNA, the “T”s would be substituted for “U”s. Thus, any of the DNAs disclosed and identified by a particular sequence identification number herein also disclose the corresponding mRNA sequence complementary to the DNA, where each “T” of the DNA sequence is substituted with “U.” An open reading frame (ORF) is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)) and ending with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA). An ORF typically encodes a protein. It will be understood that the sequences disclosed herein may further comprise additional elements, e.g., 5′ and 3′ UTRs, but that those elements, unlike the ORF, need not necessarily be present in an RNA polynucleotide of the present disclosure. Variants In some embodiments, the compositions of the present disclosure include RNA that encodes virus antigens (e.g., MV and/or EV antigens) and structurally altered variants representing a plurality of virus antigens. Antigenic variants or structurally altered variants refers to molecules that differ in their amino acid sequence from a wild-type (naturally occurring), native, or reference protein sequence. The antigen/ structurally altered variants may possess
substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants possess at least 50% identity to a wild-type, native or reference sequence. In some embodiments, variants share at least 80%, or at least 90% identity with a wild-type, native, or reference sequence. Variant antigens/polypeptides encoded by nucleic acids of the disclosure may contain amino acid changes that confer any of a number of desirable properties, e.g., that enhance their immunogenicity, vary the breadth of their immunogenicity, i.e. with respect to breadth of immune response generated, enhance their expression, and/or improve their stability or PK/PD properties in a subject. Variant antigens/polypeptides can be made using routine mutagenesis techniques and assayed as appropriate to determine whether they possess the desired property. Assays to determine expression levels and immunogenicity are well known in the art and exemplary such assays are set forth in the Examples section. Similarly, PK/PD properties of a protein variant can be measured using art recognized techniques, e.g., by determining expression of antigens in a vaccinated subject over time and/or by looking at the durability of the induced immune response. The stability of protein(s) encoded by a variant nucleic acid may be measured by assaying thermal stability or stability upon urea denaturation or may be measured using in silico prediction. Methods for such experiments and in silico determinations are known in the art. In some embodiments, a composition comprises an RNA or an RNA ORF that comprises a nucleotide sequence of any one of the sequences provided herein, or comprises a nucleotide sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a nucleotide sequence of a wild-type (naturally occurring) or variant antigen. The term “identity” refers to a relationship between the sequences of two or more polypeptides (e.g. antigens) or polynucleotides (nucleic acids), as determined by comparing the sequences. Identity also refers to the degree of sequence relatedness between or among sequences as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related antigens or nucleic acids can be readily calculated by known methods. “Percent (%) identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. It is understood that identity depends on a calculation of percent identity but may differ in
value due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide (e.g., antigen) have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res.25:3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147:195-197). A general global alignment technique based on dynamic programming is the Needleman–Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol.48:443-453). More recently a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman– Wunsch algorithm. As such, polynucleotides encoding proteins or glycoproteins containing substitutions, insertions and/or additions, deletions, and covalent modifications with respect to reference sequences, in particular the polypeptide (e.g., antigen) sequences disclosed herein, are included within the scope of this disclosure. For example, sequence tags or amino acids, such as one or more lysines, can be added to peptide sequences (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide detection, purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble or linked to a solid support. In some embodiments, sequences for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (such as, e.g., foldon regions) and the like may be substituted with alternative sequences that achieve the same or a similar function. In some embodiments, cavities in the core of proteins can be filled to improve stability, e.g., by introducing larger amino acids. In other embodiments, buried hydrogen bond networks may be replaced with hydrophobic resides to improve stability. In yet other embodiments, glycosylation sites may be removed and replaced with appropriate residues. Such sequences are readily identifiable to one of skill in the art. It should also be
understood that some of the sequences provided herein contain sequence tags or terminal peptide sequences (e.g., at the N-terminal or C-terminal ends) that may be deleted, for example, prior to use in the preparation of an mRNA vaccine. As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of orthopoxvirus antigens of interest. For example, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference antigen sequence but otherwise identical) of a reference protein, provided that the fragment is immunogenic and confers a protective immune response to an orthopoxvirus (e.g., smallpox or monkeypox). In addition to structurally altered variants that are identical to the reference protein but are truncated, in some embodiments, a structurally altered variant includes an antigen that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations with respect to a reference antigen. Some examples of structurally altered variants are shown in the sequences provided or referenced herein. Antigens/antigenic polypeptides can range in length from about 4, 6, or 8 amino acids to full length proteins. Stabilizing Elements Naturally-occurring eukaryotic mRNA molecules can contain stabilizing elements, including, but not limited to untranslated regions (UTR) at their 5′-end (5′ UTR) and/or at their 3′-end (3′ UTR), in addition to other structural features, such as a 5′-cap structure or a 3′-poly(A) tail. Both the 5′ UTR and the 3′ UTR are typically transcribed from the genomic DNA and are elements of the premature mRNA. Characteristic structural features of mature mRNA, such as the 5′-cap and the 3′-poly(A) tail are usually added to the transcribed (premature) mRNA during mRNA processing. In some embodiments, a composition includes an RNA polynucleotide having an open reading frame encoding at least one antigenic polypeptide having at least one modification, at least one 5′ terminal cap, and is formulated within a lipid nanoparticle.5′-capping of polynucleotides may be completed concomitantly during the in vitro-transcription reaction using the following chemical RNA cap analogs to generate the 5′-guanosine cap structure according to manufacturer protocols: 3´-O-Me-m7G(5')ppp(5') G [the ARCA cap];G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA).5′- capping of modified RNA may be completed post-transcriptionally using a Vaccinia Virus Capping Enzyme to generate the “Cap 0” structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2′-O methyl-transferase to generate: m7G(5')ppp(5')G-2′-O-methyl. Cap 2 structure may be
generated from the Cap 1 structure followed by the 2′-O-methylation of the 5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-O methyl-transferase. Enzymes may be derived from a recombinant source. The 3′-poly(A) tail is typically a stretch of adenine nucleotides added to the 3′-end of the transcribed mRNA. It can, in some instances, comprise up to about 400 adenine nucleotides. In some embodiments, the length of the 3′-poly(A) tail may be an essential element with respect to the stability of the individual mRNA. In some embodiments, the vaccine (e.g., multivalent RNA composition) comprises greater than 20%, 30%, 40%, 50%, or 60% polyA-tailed RNAs. In some embodiments, a composition includes a stabilizing element. Stabilizing elements may include for instance a histone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa protein has been identified. It is associated with the histone stem-loop at the 3'-end of the histone messages in both the nucleus and the cytoplasm. Its expression level is regulated by the cell cycle; it peaks during the S-phase, when histone mRNA levels are also elevated. The protein has been shown to be essential for efficient 3'-end processing of histone pre-mRNA by the U7 snRNP. SLBP continues to be associated with the stem-loop after processing, and then stimulates the translation of mature histone mRNAs into histone proteins in the cytoplasm. The RNA binding domain of SLBP is conserved through metazoa and protozoa; its binding to the histone stem-loop depends on the structure of the loop. The minimum binding site includes at least three nucleotides 5’ and two nucleotides 3′ relative to the stem-loop. In some embodiments, an mRNA includes a coding region, at least one histone stem- loop, and optionally, a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal generally should enhance the expression level of the encoded protein. The encoded protein, in some embodiments, is not a histone protein, a reporter protein (e.g. Luciferase, GFP, EGFP, β-Galactosidase, EGFP), or a marker or selection protein (e.g. alpha- Globin, Galactokinase and Xanthine:guanine phosphoribosyl transferase (GPT)). In some embodiments, an mRNA includes the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop, even though both represent alternative mechanisms in nature, acts synergistically to increase the protein expression beyond the level observed with either of the individual elements. The synergistic effect of the combination of poly(A) and at least one histone stem-loop does not depend on the order of the elements or the length of the poly(A) sequence. In some embodiments, an mRNA does not include a histone downstream element (HDE). “Histone downstream element” (HDE) includes a purine-rich polynucleotide stretch of approximately 15 to 20 nucleotides 3′ of naturally occurring stem-loops, representing the binding
site for the U7 snRNA, which is involved in processing of histone pre-mRNA into mature histone mRNA. In some embodiments, the nucleic acid does not include an intron. An mRNA may or may not contain an enhancer and/or promoter sequence, which may be modified or unmodified or which may be activated or inactivated. In some embodiments, the histone stem-loop is generally derived from histone genes and includes an intramolecular base pairing of two neighbored partially or entirely reverse complementary sequences separated by a spacer, consisting of a short sequence, which forms the loop of the structure. The unpaired loop region is typically unable to base pair with either of the stem loop elements. It occurs more often in RNA, as is a key component of many RNA secondary structures but may be present in single- stranded DNA as well. Stability of the stem-loop structure generally depends on the length, number of mismatches or bulges, and base composition of the paired region. In some embodiments, wobble base pairing (non-Watson-Crick base pairing) may result. In some embodiments, the at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides. In some embodiments, an mRNA has one or more AU-rich sequences removed. These sequences, sometimes referred to as AURES are destabilizing sequences found in the 3’UTR. The AURES may be removed from the RNA vaccines. Alternatively, the AURES may remain in the RNA vaccine. Signal Peptides In some embodiments, a composition comprises an mRNA having an ORF that encodes a signal peptide fused to an orthopoxvirus antigen. Signal peptides, comprising the N-terminal 15- 60 amino acids of proteins, are typically needed for the translocation across the membrane on the secretory pathway and, thus, universally control the entry of most proteins both in eukaryotes and prokaryotes to the secretory pathway. In eukaryotes, the signal peptide of a nascent precursor protein (pre-protein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it for processing. ER processing produces mature proteins, wherein the signal peptide is cleaved from precursor proteins, typically by an ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor. A signal peptide may also facilitate the targeting of the protein to the cell membrane. A signal peptide may have a length of 15-60 amino acids. For example, a signal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. In some embodiments, a signal peptide has a length of 20-60, 25-60, 30-60, 35-
60, 40-60, 45- 60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20- 40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15-20 amino acids. Signal peptides from heterologous genes (which regulate expression of genes other than orthopoxvirus antigens in nature) are known in the art and can be tested for desired properties and then incorporated into a nucleic acid of the disclosure. Fusion Proteins In some embodiments, a composition of the present disclosure includes an mRNA encoding an antigenic fusion protein. Thus, the encoded antigen or antigens may include two or more proteins (e.g., protein and/or protein fragment) joined together. Alternatively, the protein to which a protein antigen is fused does not promote a strong immune response to itself, but rather to the orthopoxvirus antigen. Antigenic fusion proteins, in some embodiments, retain the functional property from each original protein. Scaffold Moieties The mRNA vaccines as provided herein, in some embodiments, encode fusion proteins that comprise orthopoxvirus antigens linked to scaffold moieties. In some embodiments, such scaffold moieties impart desired properties to an antigen encoded by a nucleic acid of the disclosure. For example, scaffold proteins may improve the immunogenicity of an antigen, e.g., by altering the structure of the antigen, altering the uptake and processing of the antigen, and/or causing the antigen to bind to a binding partner. In some embodiments, the scaffold moiety is protein that can self-assemble into protein nanoparticles that are highly symmetric, stable, and structurally organized, with diameters of 10– 150 nm, a highly suitable size range for optimal interactions with various cells of the immune system. In some embodiments, viral proteins or virus-like particles can be used to form stable nanoparticle structures. Examples of such viral proteins are known in the art. For example, in some embodiments, the scaffold moiety is a hepatitis B surface antigen (HBsAg). HBsAg forms spherical particles with an average diameter of ~22 nm and which lacked nucleic acid and hence are non-infectious (Lopez-Sagaseta, J. et al. Computational and Structural Biotechnology Journal 14 (2016) 58–68). In some embodiments, the scaffold moiety is a hepatitis B core antigen (HBcAg) self-assembles into particles of 24–31 nm diameter, which resembled the viral cores obtained from HBV-infected human liver. HBcAg produced in self-assembles into two classes of differently sized nanoparticles of 300 Å and 360 Å diameter, corresponding to 180 or 240
protomers. In some embodiments, the orthopoxvirus antigen is fused to HBsAG or HBcAG to facilitate self-assembly of nanoparticles displaying the orthopoxvirus antigen. In some embodiments, bacterial protein platforms may be used. Non-limiting examples of these self-assembling proteins include ferritin, lumazine and encapsulin. Ferritin is a protein whose main function is intracellular iron storage. Ferritin is made of 24 subunits, each composed of a four-alpha-helix bundle, that self-assemble in a quaternary structure with octahedral symmetry (Cho K.J. et al. J Mol Biol.2009;390:83–98). Several high- resolution structures of ferritin have been determined, confirming that Helicobacter pylori ferritin is made of 24 identical protomers, whereas in animals, there are ferritin light and heavy chains that can assemble alone or combine with different ratios into particles of 24 subunits (Granier T. et al. J Biol Inorg Chem.2003;8:105–111; Lawson D.M. et al. Nature.1991;349:541–544). Ferritin self-assembles into nanoparticles with robust thermal and chemical stability. Thus, the ferritin nanoparticle is well-suited to carry and expose antigens. Lumazine synthase (LS) is also well-suited as a nanoparticle platform for antigen display. LS, which is responsible for the penultimate catalytic step in the biosynthesis of riboflavin, is an enzyme present in a broad variety of organisms, including archaea, bacteria, fungi, plants, and eubacteria (Weber S.E. Flavins and Flavoproteins. Methods and Protocols, Series: Methods in Molecular Biology.2014). The LS monomer is 150 amino acids long and consists of beta-sheets along with tandem alpha-helices flanking its sides. A number of different quaternary structures have been reported for LS, illustrating its morphological versatility: from homopentamers up to symmetrical assemblies of 12 pentamers forming capsids of 150 Å diameter. Even LS cages of more than 100 subunits have been described (Zhang X. et al. J Mol Biol.2006;362:753–770). Encapsulin, a novel protein cage nanoparticle isolated from thermophile Thermotoga maritima, may also be used as a platform to present antigens on the surface of self-assembling nanoparticles. Encapsulin is assembled from 60 copies of identical 31 kDa monomers having a thin and icosahedral T = 1 symmetric cage structure with interior and exterior diameters of 20 and 24 nm, respectively (Sutter M. et al. Nat Struct Mol Biol.2008, 15: 939-947). Although the exact function of encapsulin in T. maritima is not clearly understood yet, its crystal structure has been recently solved and its function was postulated as a cellular compartment that encapsulates proteins such as DyP (Dye decolorizing peroxidase) and Flp (Ferritin like protein), which are involved in oxidative stress responses (Rahmanpour R. et al. FEBS J.2013, 280: 2097-2104). In some embodiments, an RNA of the present disclosure encodes an orthopoxvirus antigen fused to a foldon domain. The foldon domain may be, for example, obtained from bacteriophage T4 fibritin (see, e.g., Tao Y, et al. Structure.1997 Jun 15; 5(6):789-98).
Linkers and Cleavable Peptides In some embodiments, the mRNAs of the disclosure encode more than one polypeptide, referred to herein as fusion proteins. In some embodiments, the mRNA further encodes a linker located between at least one or each domain of the fusion protein. The linker can be, for example, a cleavable linker or protease-sensitive linker. In some embodiments, the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, E2A linker, and combinations thereof. This family of self-cleaving peptide linkers, referred to as 2A peptides, has been described in the art (see for example, Kim, J.H. et al. (2011) PLoS ONE 6:e18556). In some embodiments, the linker is an F2A linker. In some embodiments, the linker is a GGGS (SEQ ID NO: 63) linker. In some embodiments, the fusion protein contains three domains with intervening linkers, having the structure: domain-linker-domain-linker-domain. Cleavable linkers known in the art may be used in connection with the disclosure. Exemplary such linkers include: F2A linkers,T2A linkers, P2A linkers, E2A linkers (See, e.g., WO2017127750). The skilled artisan will appreciate that other art-recognized linkers may be suitable for use in the constructs of the disclosure (e.g., encoded by the nucleic acids of the disclosure). The skilled artisan will likewise appreciate that other polycistronic constructs (mRNA encoding more than one antigen/polypeptide separately within the same molecule) may be suitable for use as provided herein. Sequence Optimization In some embodiments, an ORF encoding an antigen of the disclosure is codon optimized. Codon optimization methods are known in the art. For example, an ORF of any one or more of the sequences provided herein may be codon optimized. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art – non- limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.
In some embodiments, a codon optimized sequence shares less than 95% sequence identity to a naturally-occurring or wild-type sequence ORF (e.g., a naturally-occurring or wild- type mRNA sequence encoding an orthopoxvirus antigen). In some embodiments, a codon optimized sequence shares less than 90% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding an orthopoxvirus antigen). In some embodiments, a codon optimized sequence shares less than 85% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding an orthopoxvirus antigen). In some embodiments, a codon optimized sequence shares less than 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding an orthopoxvirus antigen). In some embodiments, a codon optimized sequence shares less than 75% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding an orthopoxvirus antigen). In some embodiments, a codon optimized sequence shares between 65% and 85% (e.g., between about 67% and about 85% or between about 67% and about 80%) sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding an orthopoxvirus antigen). In some embodiments, a codon optimized sequence shares between 65% and 75% or about 80% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding an orthopoxvirus antigen). In some embodiments, a codon-optimized sequence encodes an antigen that is as immunogenic as, or more immunogenic than (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% more), than an orthopoxvirus antigen encoded by a non-codon-optimized sequence. When transfected into mammalian host cells, the modified mRNAs have a stability of between 12-18 hours, or greater than 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours and are capable of being expressed by the mammalian host cells. In some embodiments, a codon optimized RNA may be one in which the levels of G/C are enhanced. The G/C-content of nucleic acid molecules (e.g., mRNA) may influence the stability of the RNA. RNA having an increased amount of guanine (G) and/or cytosine (C) residues may be functionally more stable than RNA containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. As an example, WO02/098443 discloses a pharmaceutical composition containing an mRNA stabilized by sequence modifications in the translated region. Due to the degeneracy of the genetic code, the modifications work by
substituting existing codons for those that promote greater RNA stability without changing the resulting amino acid. The approach is limited to coding regions of the RNA. Chemically Unmodified Nucleotides In some embodiments, an mRNA is not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g. A, G, C, or U). In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g. dA, dG, dC, or dT). Chemical Modifications The compositions of the present disclosure comprise, in some embodiments, an RNA having an open reading frame encoding an orthopoxvirus antigen, wherein the nucleic acid comprises nucleotides and/or nucleosides that can be standard (unmodified) or modified as is known in the art. In some embodiments, nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally-occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and/or nucleoside as are recognized in the art. In some embodiments, a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database. In some embodiments, a non-naturally occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such non-naturally occurring modified nucleotides and nucleosides can be found, inter alia, in published US application Nos. PCT/US2012/058519; PCT/US2013/075177; PCT/US2014/058897; PCT/US2014/058891; PCT/US2014/070413; PCT/US2015/036773; PCT/US2015/036759; PCT/US2015/036771; or PCT/IB2017/051367 all of which are incorporated by reference herein. Hence, nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) can comprise standard nucleotides and nucleosides, naturally- occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof.
Nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids), in some embodiments, comprise various (more than one) different types of standard and/or modified nucleotides and nucleosides. In some embodiments, a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and/or modified nucleotides and nucleosides. In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides. In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides. Nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the nucleic acids to achieve desired functions or properties. The modifications may be present on internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a nucleic acid may be chemically modified. The present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., RNA nucleic acids, such as mRNA nucleic acids). A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A “nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides. Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard
base structures, such as, for example, in those nucleic acids having at least one chemical modification. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base/sugar or linker may be incorporated into nucleic acids of the present disclosure. In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine (ψ). In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, and/or 5-methoxy cytidine. In some embodiments, the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications. In some embodiments, a mRNA of the disclosure comprises 1-methyl-pseudouridine (m1ψ) substitutions at one or more or all uridine positions of the nucleic acid. In some embodiments, a mRNA of the disclosure comprises 1-methyl-pseudouridine (m1ψ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid. In some embodiments, a mRNA of the disclosure comprises pseudouridine (ψ) substitutions at one or more or all uridine positions of the nucleic acid. In some embodiments, a mRNA of the disclosure comprises pseudouridine (ψ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid. In some embodiments, a mRNA of the disclosure comprises uridine at one or more or all uridine positions of the nucleic acid. In some embodiments, mRNAs are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a nucleic acid can be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above. The nucleic acids of the present disclosure may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a nucleic acid of the disclosure, or in a predetermined sequence region thereof (e.g., in the mRNA including or excluding the poly(A) tail). In some embodiments, all nucleotides X in a nucleic
acid of the present disclosure (or in a sequence region thereof) are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C. The nucleic acid may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C. The mRNAs may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the nucleic acids may contain a modified pyrimidine such as a modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the nucleic acid is replaced with a modified uracil (e.g., a 5-substituted uracil). The modified uracil can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the nucleic acid is replaced with a modified cytosine (e.g., a 5-substituted cytosine). The modified cytosine can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). Untranslated Regions (UTRs) The mRNAs of the present disclosure may comprise one or more regions or parts which act or function as an untranslated region. Where mRNAs are designed to encode at least one antigen of interest, the nucleic may comprise one or more of these untranslated regions (UTRs).
Wild-type untranslated regions of a nucleic acid are transcribed but not translated. In mRNA, the 5′ UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas the 3′ UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into the polynucleotides of the present disclosure to, among other things, enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites. A variety of 5’UTR and 3’UTR sequences are known and available in the art. A 5 ^ UTR is region of an mRNA that is directly upstream (5 ^) from the start codon (the first codon of an mRNA transcript translated by a ribosome). A 5 ^ UTR does not encode a protein (is non-coding). Natural 5′UTRs have features that play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'.5′UTR also have been known to form secondary structures which are involved in elongation factor binding. In some embodiments of the disclosure, a 5’ UTR is a heterologous UTR, i.e., is a UTR found in nature associated with a different ORF. In another embodiment, a 5’ UTR is a synthetic UTR, i.e., does not occur in nature. Synthetic UTRs include UTRs that have been mutated to improve their properties, e.g., which increase gene expression as well as those which are completely synthetic. Exemplary 5’ UTRs include Xenopus or human derived a-globin or b- globin (8278063; 9012219), human cytochrome b-245 a polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus (US8278063, 9012219). CMV immediate-early 1 (IE1) gene (US20140206753, WO2013/185069), the sequence GGGAUCCUACC (SEQ ID NO: 6) (WO2014144196) may also be used. In another embodiment, 5' UTR of a TOP gene is a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract) (e.g., WO/2015101414, WO2015101415, WO/2015/062738, WO2015024667, WO2015024667; 5' UTR element derived from ribosomal protein Large 32 (L32) gene (WO/2015101414, WO2015101415, WO/2015/062738), 5' UTR element derived from the 5'UTR of an hydroxysteroid (17-β) dehydrogenase 4 gene (HSD17B4) (WO2015024667), or a 5' UTR element derived from the 5' UTR of ATP5A1 (WO2015024667) can be used. In some embodiments, an internal ribosome entry site (IRES) is used instead of a 5' UTR.
In some embodiments, a 5' UTR of the present disclosure comprises a sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2. A 3 ^ UTR is region of an mRNA that is directly downstream (3 ^) from the stop codon (the codon of an mRNA transcript that signals a termination of translation). A 3 ^ UTR does not encode a protein (is non-coding). Natural or wild type 3′ UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3′ UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo. Introduction, removal or modification of 3′ UTR AU rich elements (AREs) can be used to modulate the stability of nucleic acids (e.g., RNA) of the disclosure. When engineering specific nucleic acids, one or more copies of an ARE can be introduced to make nucleic acids of the disclosure less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using nucleic acids of the disclosure and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection. Those of ordinary skill in the art will understand that 5’UTRs that are heterologous or synthetic may be used with any desired 3’ UTR sequence. For example, a heterologous 5’UTR may be used with a synthetic 3’UTR with a heterologous 3’ UTR. Non-UTR sequences may also be used as regions or subregions within a nucleic acid. For example, introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure. Incorporation of intronic sequences may increase protein production as well as nucleic acid levels.
Combinations of features may be included in flanking regions and may be contained within other features. For example, the ORF may be flanked by a 5' UTR which may contain a strong Kozak translational initiation signal and/or a 3' UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail.5′ UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes such as the 5′ UTRs described in US Patent Application Publication No.20100293625 and PCT/US2014/069155, herein incorporated by reference in its entirety. It should be understood that any UTR from any gene may be incorporated into the regions of a nucleic acid. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5′ or 3′ UTR may be inverted, shortened, lengthened, made with one or more other 5′ UTRs or 3′ UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3′ UTR or 5′ UTR may be altered relative to a wild-type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3′ or 5′) comprise a variant UTR. In some embodiments, a double, triple or quadruple UTR such as a 5′ UTR or 3′ UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. For example, a double beta-globin 3′ UTR may be used as described in US Patent publication 20100129877, the contents of which are incorporated herein by reference in its entirety. It is also within the scope of the present disclosure to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level. In some embodiments, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature or property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more
polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern. The untranslated region may also include translation enhancer elements (TEE). As a non- limiting example, the TEE may include those described in US Application No.20090226470, herein incorporated by reference in its entirety, and those known in the art. In vitro Transcription of RNA Aspects of the present disclosure provide methods of producing (e.g., synthesizing) a RNA transcript (e.g., mRNA transcript) comprising contacting a DNA template (e.g., a first input DNA and a second input DNA) with a RNA polymerase (e.g., a T7 RNA polymerase, a T7 RNA polymerase variant, etc.) under conditions that result in the production of the RNA transcript. This process is referred to as “in vitro transcription” or “IVT”. IVT conditions typically require a purified linear DNA template containing a promoter, nucleoside triphosphates, a buffer system that includes dithiothreitol (DTT) and magnesium ions, and a RNA polymerase. The exact conditions used in the transcription reaction depend on the amount of RNA needed for a specific application. Typical IVT reactions are performed by incubating a DNA template with a RNA polymerase and nucleoside triphosphates, including GTP, ATP, CTP, and UTP (or nucleotide analogs) in a transcription buffer. A RNA transcript having a 5 ^ terminal guanosine triphosphate is produced from this reaction. In some embodiments, a wild-type T7 polymerase is used in an IVT reaction. In some embodiments, a modified or mutant T7 polymerase is used in an IVT reaction. In some embodiments, a T7 RNA polymerase variant comprises an amino acid sequence that shares at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% identity with a wild-type T7 (WT T7) polymerase. In some embodiments, the T7 polymerase variant is a T7 polymerase variant described by International Application Publication Number WO2019/036682 or WO2020/172239, the entire contents of each of which are incorporated herein by reference. In some embodiments, the RNA polymerase (e.g., T7 RNA polymerase or T7 RNA polymerase variant) is present in a reaction (e.g., an IVT reaction) at a concentration of 0.01 mg/ml to 1 mg/ml. For example, the RNA polymerase may be present in a reaction at a concentration of 0.01 mg/mL, 0.05 mg/ml, 0.1 mg/ml, 0.5 mg/ml or 1.0 mg/ml. The input deoxyribonucleic acid (DNA) serves as a nucleic acid template for RNA polymerase. A DNA template may include a polynucleotide encoding a polypeptide of interest (e.g., an antigenic polypeptide). A DNA template, in some embodiments, includes a RNA polymerase promoter (e.g., a T7 RNA polymerase promoter) located 5’ from and operably linked to polynucleotide encoding a polypeptide of interest. A DNA template may also include a
nucleotide sequence encoding a polyadenylation (polyA) tail located at the 3’ end of the gene of interest. In some embodiments, an input DNA comprises plasmid DNA (pDNA). As used herein, “plasmid DNA” or “pDNA” refers to an extrachromosomal DNA molecule that is physically separated from chromosomal DNA in a cell and can replicate independently. In some embodiments, plasmid DNA is isolated from a cell (e.g., as a plasmid DNA preparation). In some embodiments, plasmid DNA comprises an origin of replication, which may contain one or more heterologous nucleic acids, for example nucleic acids encoding therapeutic proteins that may serve as a template for RNA polymerase. Plasmid DNA may be circularized or linear (e.g., plasmid DNA that has been linearized by a restriction enzyme digest). Multivalent mRNA constructs are typically produced by transcribing one mRNA product at a time, purifying each mRNA product, and then mixing the purified mRNA products together prior to formulation. This type of process incurs significant time and monetary investment especially at the Good Manufacturing Practice (GMP) scale. Aspects of the disclosure relate to methods for producing compositions comprising multivalent different RNAs (e.g., 2 or more different RNAs). In some aspects, methods of multivalent transcription disclosed herein involve selecting amounts of input DNA for IVT reactions that result in multivalent RNA compositions having higher purity than RNA compositions produced using previous methods. It was observed that certain characteristics or properties of DNA molecules being co-transcribed (e.g., transcribed simultaneously in vitro), such as differences in length between DNA molecules, polyA-tailing efficiency of DNA molecules, etc., and/or other reagents present in the co-IVT reaction mixture (e.g., RNA polymerase, nucleotide triphosphates (NTPs), etc.) may introduce compositional bias into the resulting multivalent RNA compositions. Surprisingly, methods were discovered that reduce such compositional bias. In some embodiments, modifying input DNA amounts results in production of multivalent RNA compositions having increased purity (e.g., as measured by percentage of RNAs comprising polyA tails) relative to RNA compositions produced by previous methods. It was also surprisingly discovered that co-IVT methods described herein result in high purity multivalent RNA compositions even when there is a large difference (e.g., >100 nucleotides) in the lengths of the input DNAs used in the IVT reaction. Accordingly, in some aspects, the disclosure provides a method for producing a multivalent RNA composition, the method comprising simultaneously in vitro transcribing at least two DNA molecules in a reaction mixture comprising: a first population of DNA molecules encoding a first RNA; a second population of DNA molecules encoding a second RNA that is different than the first RNA; and obtaining a multivalent RNA composition having a pre-defined ratio of the first RNA to the second RNA produced by the IVT.
As used herein, the term “multivalent RNA composition” refers to a composition comprising more than two different mRNAs. A multivalent RNA composition may comprise 2 or more different RNAs, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different RNAs. In some embodiments, a multivalent RNA composition comprises more than 10 different RNAs. The term “different RNAs” refers to any RNA that is not the same as another RNA in a multivalent RNA composition. For example, two RNAs are different if they have i) different lengths (whether or not the RNAs are identical over the entirety of the shorter of the two lengths), ii) different nucleotide sequences, iii) different chemical modification patterns, or iv) any combination of the foregoing. In some embodiments, each input DNA (e.g., population of input DNA molecules) in a co-IVT reaction is obtained from a different source (e.g., synthesized separately, for example in different cells or populations of cells). In some embodiments, each input DNA (e.g., population of input DNA) is obtained from a different bacterial cell or population of bacterial cells. For example, in a co-IVT reaction having three populations of input DNAs, the first input DNA is produced in bacterial cell population A, the second input DNA is produced in bacterial cell population B, and the third input DNA is produced in bacterial population C, where each of A, B, and C are not the same bacterial culture (e.g., co-cultured in the same container or plate). Methods of obtaining populations of input DNAs (e.g., plasmid DNAs) are known, for example as described by Sambrook, Joseph. Molecular Cloning : a Laboratory Manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, 2001. Some aspects comprise normalizing the amount of DNA used in the multivalent co-IVT reaction. In some embodiments, the normalization is based on the molar mass of the input DNAs. In some embodiments, the normalization is based on the degradation rate of the input DNAs. In some embodiments, the normalization is based on the degradation rate of the resultant mRNAs (e.g., measured based upon polyA variants present in the reaction mixture, or T7 polymerase abortive transcripts or truncated transcripts). In some embodiments, the normalization is based on the nucleotide content (e.g., amount of A, G, C, U, or any combination thereof) of the input DNAs. In some embodiments, the normalization is based on the purity of the input DNAs. In some embodiments the normalization is based on the polyA-tailing efficiency of the input DNAs. In some embodiments, the normalization is based on the lengths of the input DNAs. In some embodiments, mRNA is at a pre-defined mRNA ratio, which may comprise a ratio between 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different RNAs (e.g., depending on the number of different RNAs in a composition). In some embodiments, a pre-defined ratio comprises a ratio between more than 10 RNAs. As used herein, a “pre-defined mRNA ratio” refers to the desired
final ratio of RNA molecules in a multivalent RNA composition. The desired final ratio of an RNA composition will depend upon the final peptide(s) or polypeptide product(s) encoded by the RNAs. For example, a multivalent RNA mixture may comprise two RNAs (e.g., a RNA encoding a first antigen and a second antigen); in this instance the desired final ratio of RNA molecules may be 1 first antigen RNA:1 second antigen RNA. In another example, a multivalent RNA composition may comprise several (e.g., 3, 4, 5, 6, 7, 8, or more) RNAs encoding different antigenic peptides (e.g., for use as a vaccine); in that instance the desired ratio may comprise between 3 and 10 RNAs (e.g., a:b:c, a:b:c:d, a:b:c:d:e, a:b:c:d:e:f, a:b:c:d:e:f:g, a:b:c:d:e:f:g:h, a:b:c:d:e:f:g:h:i, a:b:c:d:e:f:g:h:i:j, etc., where each of a-j is a number between 1 and 10). In some embodiments, the normalization is based on the lowest level present in the input DNAs (e.g., lowest molar mass, degradation rate (e.g., of the input DNA and/or output RNA), nucleotide content, purity, and/or polyA-tailing efficiency). In some embodiments, the normalization is based on the highest level present in the input DNAs (e.g., highest molar mass, degradation rate (e.g., of the input DNA and/or output RNA), nucleotide context, purity, and/or polyA-tailing efficiency). In some embodiments, the normalization is based on the rate of RNA production of the input DNAs (e.g., the highest rate of RNA production of an input DNA or the lowest rate of RNA production of an input DNA in a reaction mixture). In some aspects, the disclosure relates to IVT methods in which the amount of input DNA (e.g., a first DNA or second DNA) is adjusted or normalized in order to improve production of multivalent RNA compositions having a pre-defined mRNA ratio of components. As described herein, certain factors affecting multivalent RNA composition purity, such as large differences in size between input DNAs (e.g., a difference of more than 100, 200, 500, 1000, or more nucleotides in length) and/or polyA-tailing efficiency of a given DNA during IVT, may be addressed prior to the IVT by normalizing the amount of input DNA based upon one or more of those factors. The number of input DNAs (e.g., populations of input DNA molecules) used in an IVT reaction may vary, depending upon the number of different RNA molecules desired to be included in the multivalent RNA composition. In some embodiments, an IVT reaction mixture comprises 2 or more different input DNAs, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more different input DNAs. In some embodiments, the IVT reaction comprises more than 15 different input DNAs. The term “different input DNAs” encompasses input DNAs that encode different RNAs, e.g., that have i) different lengths (whether or not the RNAs are identical over the entirety of the shorter of the two lengths), ii) different nucleotide sequences, iii) different chemical modification patterns, or iv) any combination of the foregoing.
In some embodiments, two or more of the input DNA molecules used in an IVT reaction encode mRNA molecules that have a different length (e.g., comprises a different number of nucleotides). In some embodiments, the difference in length between two or more of the mRNA molecules encoded by different input DNA molecules in an IVT reaction mixture is greater than 70 nucleotides, 80 nucleotides, 90 nucleotides, or 100 nucleotides (e.g., two input DNAs in a composition encode mRNA molecules that are not are within 70, 80, 90, or 100 nucleotides in length of one another). In some embodiments, the difference in length between two or more of the mRNA molecules encoded by different input DNA molecules is more than 100 nucleotides, for example 500 nucleotides, 1000 nucleotides, 1500 nucleotides, 2000 nucleotides, 3000 nucleotides, 4000 nucleotides, or more. In specific embodiments, the vaccine (e.g., multivalent RNA composition) is produced by combining a linearized first DNA molecule encoding the first mRNA polynucleotide, a linearized second DNA molecule encoding the second mRNA polynucleotide, and a linearized third DNA molecule encoding the third mRNA polynucleotide into a single reaction vessel, wherein the first DNA molecule, the second DNA molecule, and the third DNA molecule are obtained from different sources. In some embodiments, the different sources are a first, second, and third bacterial cell culture and wherein the first, second and third bacterial cell culture are not co-cultured. In some embodiments, the different sources are a first, second, and third bacterial cell culture and wherein the first, second and third bacterial cell culture are co-cultured. In some embodiments, the amounts of the first, second and third DNA molecules present in the reaction mixture prior to the start of the in vitro transcription have been normalized. In some embodiments, the linearized first DNA molecule, the linearized second DNA molecule and the linearized third DNA molecule are simultaneously in vitro transcribed to obtain the multivalent RNA composition. In some embodiments, an in vitro transcription template encodes a 5′ untranslated (UTR) region, contains an open reading frame, and encodes a 3′ UTR and a poly(A) tail. The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template. A “5′ untranslated region” (UTR) refers to a region of an mRNA that is directly upstream (i.e., 5′) from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide. When RNA transcripts are being generated, the 5’ UTR may comprise a promoter sequence. Such promoter sequences are known in the art. It should be understood that such promoter sequences will not be present in a vaccine of the disclosure.
A “3′ untranslated region” (UTR) refers to a region of an mRNA that is directly downstream (i.e., 3′) from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide. An “open reading frame” is a continuous stretch of DNA beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a polypeptide. A “poly(A) tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3′), from the 3′ UTR that contains multiple, consecutive adenosine monophosphates. A poly(A) tail may contain 10 to 300 adenosine monophosphates. For example, a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a poly(A) tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo) the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, and/or export of the mRNA from the nucleus and translation. In some embodiments, a nucleic acid includes 200 to 3,000 nucleotides. For example, a nucleic acid may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides). An in vitro transcription system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs. Any number of RNA polymerases or variants may be used in the method of the present disclosure. The polymerase may be selected from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and/or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids and/or modified nucleotides, including chemically modified nucleic acids and/or nucleotides. Some embodiments exclude the use of DNase. In some embodiments, the RNA transcript is capped via enzymatic capping. In some embodiments, the RNA comprises 5' terminal cap, for example, 7mG(5’)ppp(5’)NlmpNp.
Non-coding Sequences Aspects of the disclosure relate to multivalent RNA compositions which comprise mRNAs, e.g., 2-15 mRNA polynucleotides each comprising a distinct open reading frame (ORF) encoding an orthopoxvirus antigenic polypeptide, wherein each mRNA polynucleotide comprises one or more non-coding sequences in an untranslated region (UTR) having unique identifier sequences or non-coding sequences. As used herein, “non-coding sequence” refers to a sequence of a biological molecule (e.g., nucleic acid, protein, etc.) that when combined with the sequence another biological molecule serves to identify the other biological molecule. Typically, a non-coding sequence is a heterologous sequence that is incorporated within or appended to a sequence of a target biological molecule and utilized as a reference in order to identify a target molecule of interest. In some embodiments, a non-coding sequence is a sequence of a nucleic acid (e.g., a heterologous or synthetic nucleic acid) that is incorporated within or appended to a target nucleic acid and utilized as a reference in order to identify the target nucleic acid. In some embodiments, a non-coding sequence is of the formula (N)n. In some embodiments, n is an integer in the range of 5 to 20, 5 to 10, 10 to 20, 7 to 20, or 7 to 30. In some embodiments, n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more. In some embodiments, N are each nucleotides that are independently selected from A, G, T, U, and C, or analogues thereof. Thus, some embodiments comprise nucleic acids (e.g., mRNAs) that (i) have a target sequence of interest (e.g., a coding sequence (e.g., that encodes therapeutic peptide or therapeutic protein)); and (ii) comprises a unique non-coding sequence. In some embodiments, one or more in vitro transcribed mRNAs comprise one or more non-coding sequences in an untranslated region (UTR), such as a 5’ UTR or 3’ UTR. Inclusion of a non-coding sequence in the UTR of an mRNA prevents non-coding sequence from being translated into a peptide. In some embodiments, a non-coding sequence is positioned in a 3’ UTR of an mRNA. In some embodiments, the non-coding sequence is positioned upstream of the polyA tail of the mRNA. In some embodiments, the non-coding sequence is positioned downstream of (e.g., after) the polyA tail of the mRNA. In some embodiments, the non-coding sequence is positioned between the last codon of the ORF of the mRNA and the first “A” of the polyA tail of the mRNA. In some embodiments, a polynucleotide non-coding sequence positioned in a UTR comprises between 1 and 10 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides). In some embodiments, UTR comprising a polynucleotide non-coding sequence further comprises one or more (e.g., 1, 2, 3, or more) RNAse cleavage sites, such as RNase H cleavage sites. In some embodiments, each different RNA of a multivalent RNA composition comprises a different (e.g., unique) non-coding sequence. In some embodiments, RNAs of a
multivalent RNA composition are detected and/or purified according to the polynucleotide non- coding sequences of the RNAs. In some embodiments, the mRNA non-coding sequences are used to identify the presence of mRNA or determine a relative ratio of different mRNAs in a sample (e.g., a reaction product or a drug product). In some embodiments, the mRNA non- coding sequences are detected using one or more of deep sequencing, PCR, and Sanger sequencing. Exemplary non-coding sequences include: AACGUGAU; AAACAUCG; ATGCCUAA; AGUGGUCA; ACCACUGU; ACAUUGGC; CAGAUCUG; CAUCAAGU; CGCUGAUC; ACAAGCUA; CUGUAGCC; AGUACAAG; AACAACCA; AACCGAGA; AACGCUUA; AAGACGGA; AAGGUACA; ACACAGAA; ACAGCAGA; ACCUCCAA; ACGCUCGA; ACGUAUCA; ACUAUGCA; AGAGUCAA; AGAUCGCA; AGCAGGAA; AGUCACUA; AUCCUGUA; AUUGAGGA; CAACCACA; GACUAGUA; CAAUGGAA; CACUUCGA; CAGCGUUA; CAUACCAA; CCAGUUCA; CCGAAGUA; ACAGUG; CGAUGU; UUAGGC; AUCACG; and UGACCA. In some embodiments the multivalent RNA composition is produced by a method comprising: (a) combining a linearized first DNA molecule encoding the first mRNA polynucleotide, a linearized second DNA molecule encoding the second mRNA polynucleotide, and a linearized third, fourth, fifth, sixth, seventh, eighth, ninth or tenth DNA molecule encoding the third, fourth, fifth, sixth, seventh, eighth, ninth or tenth mRNA polynucleotide into a single reaction vessel, wherein the first DNA molecule, the second DNA molecule, and the third, fourth, fifth, sixth, seventh, eighth, ninth or tenth DNA molecule are obtained from different sources; and (b) simultaneously in vitro transcribing the linearized first DNA molecule, the linearized second DNA molecule and the linearized third, fourth, fifth, sixth, seventh, eighth, ninth or tenth DNA molecule to obtain a multivalent RNA composition. The different sources may be bacterial cell cultures which may not be co-cultured. In some embodiments the amounts of the first, second and third, fourth, fifth, sixth, seventh, eighth, ninth or tenth DNA molecules present in the reaction mixture prior to the start of the IVT have been normalized. Chemical Synthesis Solid-phase chemical synthesis. Nucleic acids the present disclosure may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Solid-phase synthesis is useful in site-specific introduction of chemical modifications in the nucleic acid sequences.
Liquid Phase Chemical Synthesis. The synthesis of nucleic acids of the present disclosure by the sequential addition of monomer building blocks may be carried out in a liquid phase. Combination of Synthetic Methods. The synthetic methods discussed above each has its own advantages and limitations. Attempts have been conducted to combine these methods to overcome the limitations. Such combinations of methods are within the scope of the present disclosure. The use of solid-phase or liquid-phase chemical synthesis in combination with enzymatic ligation provides an efficient way to generate long chain nucleic acids that cannot be obtained by chemical synthesis alone. Ligation of Nucleic Acid Regions or Subregions Assembling nucleic acids by a ligase may also be used. DNA or RNA ligases promote intermolecular ligation of the 5’ and 3’ ends of polynucleotide chains through the formation of a phosphodiester bond. Nucleic acids such as chimeric polynucleotides and/or circular nucleic acids may be prepared by ligation of one or more regions or subregions. DNA fragments can be joined by a ligase catalyzed reaction to create recombinant DNA with different functions. Two oligodeoxynucleotides, one with a 5’ phosphoryl group and another with a free 3’ hydroxyl group, serve as substrates for a DNA ligase. Purification Purification of the nucleic acids described herein may include, but is not limited to, nucleic acid clean-up, quality assurance and quality control. Clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term “purified” when used in relation to a nucleic acid such as a “purified nucleic acid” refers to one that is separated from at least one contaminant. A “contaminant” is any substance that makes another unfit, impure or inferior. Thus, a purified nucleic acid (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method. A quality assurance and/or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC. In some embodiments, the nucleic acids may be sequenced by methods including, but not limited to reverse-transcriptase-PCR.
Quantification In some embodiments, the nucleic acids of the present disclosure may be quantified in exosomes or when derived from one or more bodily fluid. Bodily fluids include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. Alternatively, exosomes may be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta. Assays may be performed using construct specific probes, cytometry, qRT-PCR, real- time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes may be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof. These methods afford the investigator the ability to monitor, in real time, the level of nucleic acids remaining or delivered. This is possible because the nucleic acids of the present disclosure, in some embodiments, differ from the endogenous forms due to the structural or chemical modifications. In some embodiments, the nucleic acid may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis). A non-limiting example of a UV/Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, MA). The quantified nucleic acid may be analyzed in order to determine if the nucleic acid may be of proper size, check that no degradation of the nucleic acid has occurred. Degradation of the nucleic acid may be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC- HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
Lipid Nanoparticles (LNPs) In some embodiments, the mRNA of the disclosure is formulated in a lipid nanoparticle (LNP). Lipid nanoparticles typically comprise ionizable amino lipid, non-cationic lipid, sterol and PEG lipid components along with the nucleic acid cargo of interest. The lipid nanoparticles of the disclosure can be generated using components, compositions, and methods as are generally known in the art, see for example PCT/US2016/052352; PCT/US2016/068300; PCT/US2017/037551; PCT/US2015/027400; PCT/US2016/047406; PCT/US2016/000129; PCT/US2016/014280; PCT/US2016/014280; PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394; PCT/US2016/052117; PCT/US2012/069610; PCT/US2017/027492; PCT/US2016/059575 and PCT/US2016/069491 all of which are incorporated by reference herein in their entirety. Vaccines of the present disclosure are typically formulated in lipid nanoparticles. The vaccines can be made, for example, using mixing processes such as microfluidics and T- junction mixing of two fluid streams, one of which contains the mRNA and the other has the lipid components. In some embodiments, the vaccines are prepared by combining an ionizable amino lipid, a phospholipid (such as DOPE or DSPC), a PEG lipid (such as 1,2-dimyristoyl-OT- glycerol methoxypoly ethylene glycol, also known as PEG-DMG), and a structural lipid (such as cholesterol) in an alcohol (e.g., ethanol). The lipids may be combined to yield desired molar ratios and diluted with water and alcohol (e.g., ethanol) to a final lipid concentration of between about 5.5 mM and about 25 mM, for example. Vaccines including mRNA and a lipid component may be prepared, for example, by combining a lipid solution with an mRNA solution at lipid component to mRNA wt:wt ratios of between about 5:1 and about 50:1. The lipid solution may be rapidly injected using a microfluidic based system (e.g., NanoAssemblr) at flow rates between about 10 ml/min and about 18 ml/min, for example, into the mRNA solution to produce a suspension (e.g., with a water to alcohol ratio between about 1:1 and about 4:1). Vaccines can be processed by dialysis to remove the alcohol (e.g., ethanol) and achieve buffer exchange. Formulations may be dialyzed against phosphate buffered saline (PBS), pH 7.4, for example, at volumes greater than that of the primary product (e.g., using Slide-A-Lyzer cassettes (Thermo Fisher Scientific Inc., Rockford, IL)) with a molecular weight cutoff of 10 kD, for example. The forgoing exemplary method induces nanoprecipitation and particle formation. Alternative processes including, but not limited to, T-junction and direct injection, may be used to achieve the same nanoprecipitation. Vaccines of the present disclosure are typically formulated in lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises at least one ionizable amino lipid, at least one
non-cationic lipid, at least one sterol, and/or at least one polyethylene glycol (PEG)-modified lipid. In some embodiments, the lipid nanoparticle comprises 20-60 mol% ionizable amino lipid. For example, the lipid nanoparticle may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% ionizable amino lipid. In some embodiments, the lipid nanoparticle comprises 20 mol%, 30 mol%, 40 mol%, 50, or 60 mol% ionizable amino lipid. In some embodiments, the lipid nanoparticle comprises 5-25 mol% non-cationic lipid. For example, the lipid nanoparticle may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% non-cationic lipid. In some embodiments, the lipid nanoparticle comprises 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% non-cationic lipid. In some embodiments, the lipid nanoparticle comprises 25-55 mol% sterol. For example, the lipid nanoparticle may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% sterol. In some embodiments, the lipid nanoparticle comprises 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% sterol. In some embodiments, the lipid nanoparticle comprises 0.5-15 mol% PEG-modified lipid. For example, the lipid nanoparticle may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol%. In some embodiments, the lipid nanoparticle comprises 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises 20-60 mol% ionizable amino lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises 40-50 mol% ionizable amino lipid, 5-15 mol% neutral lipid, 20-40 mol% cholesterol, and 0.5-3 mol% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises 45-50 mol% ionizable amino lipid, 9-13 mol% neutral lipid, 35-45 mol% cholesterol, and 2-3 mol% PEG-modified lipid. In some embodiments, the lipid nanoparticle comprises 48 mol% ionizable amino lipid, 11 mol% neutral lipid, 68.5 mol% cholesterol, and 2.5 mol% PEG-modified lipid. In some embodiments, an ionizable amino lipid of the disclosure comprises a compound of Formula (I):
or a salt or isomer thereof, wherein: R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and –C(R)N(R)2C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycle and heterocycle; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H;
each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13. In some embodiments, a subset of compounds of Formula (I) includes those in which when R4 is -(CH2)nQ, -(CH2)nCHQR, –CHQR, or -CQ(R)2, then (i) Q is not -N(R)2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2. In some embodiments, another subset of compounds of Formula (I) includes those in which R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and a 5- to 14-membered heterocycloalkyl having one or more heteroatoms selected from N, O, and S which is substituted with one or more substituents selected from oxo (=O), OH, amino, mono- or di-alkylamino, and C1-3 alkyl, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group;
R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycle and heterocycle; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof. In some embodiments, another subset of compounds of Formula (I) includes those in which R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(=NR9)N(R)2, and each n is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5- to 14-membered heterocycle and (i) R4 is -(CH2)nQ in which n is 1 or 2, or (ii) R4 is -(CH2)nCHQR in which n is 1, or (iii) R4 is -CHQR, and -CQ(R)2, then Q is either a 5- to 14-membered heteroaryl or 8- to 14-membered heterocycloalkyl;
each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycle and heterocycle; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof. In some embodiments, another subset of compounds of Formula (I) includes those in which R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2)nOR,
-N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(=NR9)N(R)2, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; R8 is selected from the group consisting of C3-6 carbocycle and heterocycle; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof. In some embodiments, another subset of compounds of Formula (I) includes those in which R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R2 and R3 are independently selected from the group consisting of H, C2-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4 is -(CH2)nQ or -(CH2)nCHQR, where Q is -N(R)2, and n is selected from 3, 4, and 5;
each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C1-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof. In some embodiments, another subset of compounds of Formula (I) includes those in which R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R2 and R3 are independently selected from the group consisting of C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4 is selected from the group consisting of -(CH2)nQ, -(CH2)nCHQR, -CHQR, and -CQ(R)2, where Q is -N(R)2, and n is selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;
M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C1-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof. In some embodiments, a subset of compounds of Formula (I) includes those of Formula (IA):
, or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M1 is a bond or M’; R4 is unsubstituted C1-3 alkyl, or -(CH2)nQ, in which Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group; and R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl. In some embodiments, a subset of compounds of Formula (I) includes those of Formula (II):
(II) or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and 5; M1 is a bond or M’; R4 is unsubstituted C1-3 alkyl, or -(CH2)nQ, in which n is 2, 3, or 4, and Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group; and R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl. In some embodiments, a subset of compounds of Formula (I) includes those of Formula (IIa), (IIb), (IIc), or (IIe):
or a salt or isomer thereof, wherein R4 is as described herein. In some embodiments, a subset of compounds of Formula (I) includes those of Formula (IId):
(IId), or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R’, R”, and R2 through R6 are as described herein. For example, each of R2 and R3 may be independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl. In some embodiments, an ionizable amino lipid of the disclosure comprises a compound having structure:
In some embodiments, an ionizable amino lipid of the disclosure comprises a compound having structure:
In some embodiments, a non-cationic lipid of the disclosure comprises 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero- phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-
phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In some embodiments, a PEG modified lipid of the disclosure comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is DMG-PEG, PEG-c- DOMG (also referred to as PEG-DOMG), PEG-DSG and/or PEG-DPG. In some embodiments, a sterol of the disclosure comprises cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha- tocopherol, and mixtures thereof. In some embodiments, a LNP of the disclosure comprises an ionizable amino lipid of Compound 1, wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG lipid is DMG-PEG (e.g., PEG2000-DMG). In some embodiments, the lipid nanoparticle comprises 45 – 55 mole percent (mol%) ionizable amino lipid (e.g., Compound 1). For example, lipid nanoparticle may comprise 45-47, 45-48, 45-49, 45-50, 45-52, 46-48, 46-49, 46-50, 46-52, 46-55, 47-48, 47-49, 47-50, 47-52, 47- 55, 48-50, 48-52, 48-55, 49-50, 49-52, 49-55, or 50-55 mol% ionizable amino lipid (e.g., Compound 1). For example, lipid nanoparticle may comprise 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% ionizable amino lipid. In some embodiments, the lipid nanoparticle comprises 5 – 15 mol% non-cationic (neutral) lipid (e.g., DSPC). For example, the lipid nanoparticle may comprise 5-6, 5-7, 5-8, 5-9, 5-10, 5-11, 5-12, 5-13, 5-14, 5-15, 6-7, 6-8, 6-9, 6-10, 6-11, 6-12, 6-13, 6-14, 6-15, 7-8, 7-9, 7- 10, 7-11, 7-12, 7-13, 7-14, 7-15, 8-9, 8-10, 8-11, 8-12, 8-13, 8-14, 8-15, 9-10, 9-11, 9-12, 9-13, 9-14, 9-15, 10-11, 10-12, 10-13, 10-14, 10-15, 11-12, 11-13, 11-14, 11-15, 12-13, 12-14, 13-14, 13-15, or 14-15 mol% non-cationic (neutral) lipid (e.g., DSPC). For example, the lipid nanoparticle may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% DSPC. In some embodiments, the lipid nanoparticle comprises 35 – 40 mol% sterol (e.g., cholesterol). For example, the lipid nanoparticle may comprise 35-36, 35-37, 35-38, 35-39, 35- 40, 36-37, 36-38, 36-39, 36-40, 37-38, 37-39, 37-40, 38-39, 38-40, or 39-40 mol% cholesterol. For example, the lipid nanoparticle may comprise 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, or 40 mol% cholesterol. In some embodiments, the lipid nanoparticle comprises 1 – 3 mol% DMG-PEG. For example, the lipid nanoparticle may comprise 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5,
2-3, or 2.5-3. mol% DMG-PEG. For example, the lipid nanoparticle may comprise 1, 1.5, 2, 2.5, or 3 mol% DMG-PEG. In some embodiments, the lipid nanoparticle comprises 50 mol% ionizable amino lipid, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG. In some embodiments, the lipid nanoparticle comprises 48 mol% ionizable amino lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% PEG2000-DMG. In some embodiments, an LNP of the disclosure comprises an N:P ratio of from about 2:1 to about 30:1. In some embodiments, an LNP of the disclosure comprises an N:P ratio of about 6:1. In some embodiments, an LNP of the disclosure comprises an N:P ratio of about 3:1. In some embodiments, an LNP of the disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of from about 10:1 to about 100:1. In some embodiments, an LNP of the disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of about 20:1. In some embodiments, an LNP of the disclosure comprises a wt/wt ratio of the ionizable amino lipid component to the RNA of about 10:1. In some embodiments, an LNP of the disclosure has a mean diameter from about 50 nm to about 150 nm. In some embodiments, an LNP of the disclosure has a mean diameter from about 70 nm to about 120 nm. Multivalent Vaccines The compositions, as provided herein, may include RNA or multiple RNAs encoding two or more antigens of the same or different species; that is, the compositions may be multivalent compositions (e.g., vaccines). In some embodiments, the composition includes an RNA or multiple RNAs encoding two or more orthopoxvirus antigens. In some embodiments, the RNA may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more orthopoxvirus antigens. In some embodiments, two or more different mRNA encoding antigens may be formulated in the same lipid nanoparticle. In other embodiments, two or more different RNA encoding antigens may be formulated in separate lipid nanoparticles (each RNA formulated in a single lipid nanoparticle). The lipid nanoparticles may then be combined and administered as a single vaccine composition (e.g., comprising multiple RNA encoding multiple antigens) or may be administered separately.
Pharmaceutical Formulations Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits and reagents for prevention or treatment of orthopoxviruses in humans and other mammals, for example. The compositions provided herein can be used as therapeutic or prophylactic agents. They may be used in medicine to prevent and/or treat an orthopoxvirus infection. In some embodiments, the orthopoxvirus vaccine containing RNA as described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the RNA polynucleotides are translated in vivo to produce an antigenic polypeptide (antigen). An “effective amount” of a composition (e.g., comprising RNA) is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the RNA (e.g., length, nucleotide composition, and/or extent of modified nucleosides), other components of the vaccine, and other determinants, such as age, body weight, height, sex and general health of the subject. Typically, an effective amount of a composition provides an induced or boosted immune response as a function of antigen production in the cells of the subject. In some embodiments, an effective amount is the amount necessary to prevent infection or reduce the severity of an orthopoxvirus infection in the subject based on a single dose of the vaccine or single dose of the vaccine with a booster dose. In some embodiments, an effective amount of the composition containing RNA polynucleotides having at least one chemical modification are more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or a peptide antigen. Increased antigen production may be demonstrated by increased cell transfection (the percentage of cells transfected with the RNA vaccine), increased protein translation and/or expression from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by increased duration of protein translation from a modified polynucleotide), or altered antigen specific immune response of the host cell. The term "pharmaceutical composition" refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. A "pharmaceutically acceptable carrier," after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be "acceptable" also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate.
Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences. In some embodiments, the compositions (comprising polynucleotides and their encoded polypeptides) in accordance with the present disclosure may be used for treatment or prevention of an orthopoxvirus infection. A composition may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase or during active infection after onset of symptoms. In some embodiments, the amount of RNA provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis. A vaccine, disclosed herein, may be administered to a subject to induce an antigen specific immune response, as a singular vaccine (i.e., where both mRNAs encoding antigens are included in the same formulation) or as separate vaccines (i.e., the mRNA encoding a first antigen and the mRNA encoding a second antigen are administered separately). When the vaccine is administered as a separate vaccine, the two mRNAs may be administered to the subject at the same time (i.e., within an hour of one another) or at different times (i.e., separated by more than an hour, 12 hours, 24 hours, 2 days, 7 days, 2 weeks). When the vaccine is administered as a separate vaccine the two mRNAs may be administered to the subject at the same site or a different site (i.e., as an injection in separate arms). In some embodiments the vaccine may be the only vaccine comprising a nucleic acid encoding an orthopoxvirus antigen that a subject receives. Alternatively, the vaccine may be administered in various combinations, as a prime and/or boost dose. The vaccine may be administered to seropositive or seronegative subjects. For example, a subject may be naïve and not have antibodies that react with a virus having an antigen, wherein the antigen is the viral antigen or fragment thereof encoded by the mRNA of the vaccine. Such a subject is said to be seronegative with respect to that vaccine. Alternatively, the subject may have preexisting antibodies to viral antigen encoded by the mRNA of the vaccine because they have previously had an infection with virus carrying the antigen or may have previously been administered a dose of a vaccine (e.g., an mRNA vaccine) that induces antibodies against the antigen. Such a subject is said to be seropositive with respect to that vaccine. In some instances the subject may have been previously exposed to a virus but not to a specific variant or strain of the virus or a specific vaccine associated with that variant or strain. Such a subject is considered to be seronegative with respect to the specific variant or strain. Thus, the present disclosure provides compositions (e.g., mRNA vaccines) that elicit potent neutralizing antibodies against orthopoxvirus antigens in a subject. Such a composition can be administered to seropositive or seronegative subjects in some embodiments. A
seronegative subject may be naïve and not have antibodies that react with the specific virus which the subject is being immunized against. A seropositive subject may have preexisting antibodies to the specific virus because they have previously had an infection with that virus, variant or strain or may have previously been administered a dose of a vaccine (e.g., an mRNA vaccine) that induces antibodies against that virus, variant or strain. In some embodiments, a composition includes mRNA encoding at least one (e.g., one, two, or more) orthopoxvirus antigens, such as monkeypox virus antigens from different monkeypox mutant strains (also referred to herein as variants). In some embodiments, the mRNA vaccine comprises multiple mRNAs encoding monkeypox antigens from different variants in a single lipid nanoparticle. A composition may be administered with other prophylactic or therapeutic compounds. As a non-limiting example, a prophylactic or therapeutic compound may be an adjuvant or a booster. As used herein, when referring to a prophylactic composition, such as a vaccine, the term “booster” or “booster vaccine” refers to an extra administration of the prophylactic vaccine composition. In some embodiments, the booster vaccine comprises at least one mRNA polynucleotide having an ORF encoding the first, second or third an orthopoxvirus antigenic polypeptides. In some embodiments, the booster vaccine comprises at least one mRNA polynucleotide having an ORF encoding each of the first, second and third an orthopoxvirus antigenic polypeptides. In some embodiments, the booster vaccine comprises at least one mRNA polynucleotide having an ORF encoding a variant of the first, second or third an orthopoxvirus antigenic polypeptides. A booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition. The time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months. In exemplary embodiments, the time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or 6 months. In one embodiment, the booster vaccine is administered between three weeks and one year after the vaccine.
In some embodiments, a composition may be administered intramuscularly, intranasally or intradermally, similarly to the administration of inactivated vaccines known in the art. A composition may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. As a non-limiting example, the RNA vaccines may be utilized to treat and/or prevent a variety of infectious disease. RNA vaccines have superior properties in that they produce much larger antibody titers, better neutralizing immunity, produce more durable immune responses, and/or produce responses earlier than commercially available vaccines. Provided herein are pharmaceutical compositions including RNA and/or complexes optionally in combination with one or more pharmaceutically acceptable excipients. The RNA may be formulated or administered alone or in conjunction with one or more other components. For example, an immunizing composition may comprise other components including, but not limited to, adjuvants. In some embodiments, an immunizing composition does not include an adjuvant (they are adjuvant free). An RNA may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients. In some embodiments, vaccine compositions comprise at least one additional active substance, such as, for example, a therapeutically-active substance, a prophylactically-active substance, or a combination of both. Vaccine compositions may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and/or manufacture of pharmaceutical agents, such as vaccine compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety). In some embodiments, an immunizing composition is administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the RNA vaccines or the polynucleotides contained therein, for example, RNA polynucleotides (e.g., mRNA polynucleotides) encoding antigens. Formulations of the vaccine compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., mRNA polynucleotide) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit. Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the
disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient. In some embodiments, an RNA is formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation); (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein (antigen) in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the RNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof. Dosing/Administration Provided herein are immunizing compositions (e.g., RNA vaccines), methods, kits and reagents for prevention and/or treatment of at least one an orthopoxvirus infection in humans and other mammals. Immunizing compositions can be used as therapeutic or prophylactic agents. In some embodiments, immunizing compositions are used to provide prophylactic protection from orthopoxvirus infections. In some embodiments, immunizing compositions are used to treat orthopoxvirus infections. In some embodiments, immunizing compositions are used to reduce the severity of an orthopoxvirus infection in a subject. In some embodiments, embodiments, immunizing compositions are used in the priming of immune effector cells, for example, to activate peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re- infused) into a subject. A subject may be any mammal, including non-human primate and human subjects. Typically, a subject is a human subject. In some embodiments, the subject is 60 years of age or older (e.g., 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 years of age or older). In some embodiments, the subject is under 18 years of age (e.g., under 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 years of age). In some embodiments, an immunizing composition (e.g., RNA a vaccine) is administered to a subject (e.g., a mammalian subject, such as a human subject) in an effective amount to induce an antigen-specific immune response. The RNA encoding the orthopoxvirus antigen is
expressed and translated in vivo to produce the antigen, which then stimulates an immune response in the subject. Prophylactic protection from an orthopoxvirus can be achieved following administration of an immunizing composition (e.g., an RNA vaccine) of the present disclosure. Immunizing compositions can be administered once, twice, three times, four times or more but it is likely sufficient to administer the vaccine once (optionally followed by a single booster). It is possible, although less desirable, to administer an immunizing composition to an infected individual to achieve a therapeutic response. Dosing may need to be adjusted accordingly. A method of eliciting an immune response in a subject against an orthopoxvirus antigen (or multiple antigens) is provided in aspects of the present disclosure. In some embodiments, a method involves administering to the subject an immunizing composition comprising a mRNA having an open reading frame encoding an orthopoxvirus antigen, thereby inducing in the subject an immune response specific to the orthopoxvirus antigen, wherein anti-antigen antibody titer in the subject is increased following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the antigen. An “anti-antigen antibody” is a serum antibody the binds specifically to the antigen. A prophylactically effective dose is an effective dose that prevents infection with the virus at a clinically acceptable level. In some embodiments, the effective dose is a dose listed in a package insert for the vaccine. A traditional vaccine, as used herein, refers to a vaccine other than the mRNA vaccines of the present disclosure. For instance, a traditional vaccine includes, but is not limited, to live microorganism vaccines, killed microorganism vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, virus like particle (VLP) vaccines, etc. In exemplary embodiments, a traditional vaccine is a vaccine that has achieved regulatory approval and/or is registered by a national drug regulatory body, for example the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA). In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log to 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the orthopoxvirus or an unvaccinated subject. In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log, 2 log, 3 log, 4 log, 5 log, or 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the orthopoxvirus or an unvaccinated subject. A method of eliciting an immune response in a subject against an orthopoxvirus is provided in other aspects of the disclosure. The method involves administering to the subject an immunizing composition (e.g., an RNA vaccine) comprising a RNA polynucleotide comprising
an open reading frame encoding an orthopoxvirus antigen, thereby inducing in the subject an immune response specific to the orthopoxvirus, wherein the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine against the orthopoxvirus at 2 times to 100 times the dosage level relative to the immunizing composition. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at twice the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at three times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 4 times, 5 times, 10 times, 50 times, or 100 times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 10 times to 1000 times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 100 times to 1000 times the dosage level relative to an immunizing composition of the present disclosure. In other embodiments, the immune response is assessed by determining [protein] antibody titer in the subject. In other embodiments, the ability to promote a robust T cell response(s) is measured using art recognized techniques. Other aspects the disclosure provide methods of eliciting an immune response in a subject against an orthopoxvirus by administering to the subject an immunizing composition (e.g., an RNA vaccine) comprising an RNA having an open reading frame encoding an orthopoxvirus antigen, thereby inducing in the subject an immune response specific to the orthopoxvirus antigen, wherein the immune response in the subject is induced 2 days to 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the orthopoxvirus. In some embodiments, the immune response in the subject is induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine at 2 times to 100 times the dosage level relative to an immunizing composition of the present disclosure. In some embodiments, the immune response in the subject is induced 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.
Also provided herein are methods of eliciting an immune response in a subject against an orthopoxvirus by administering to the subject an RNA having an open reading frame encoding a first antigen, wherein the RNA does not include a stabilization element, and wherein an adjuvant is not co-formulated or co-administered with the vaccine. An immunizing composition (e.g., an RNA vaccine) may be administered by any route that results in a therapeutically effective outcome. These include, but are not limited, to intradermal, intramuscular, intranasal, and/or subcutaneous administration. The present disclosure provides methods comprising administering RNA vaccines to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The RNA is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the RNA may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. The effective amount of the RNA, as provided herein, may be as low as 25 µg (total mRNA), administered for example as a single dose or as two 12.5 µg doses. A “dose” as used herein, represents the sum total of RNA in the composition (e.g., including all of the NA antigens and/or HA antigens in the formulation). In some embodiments, the effective amount is a total dose of 25 µg-300 µg, 50 µg-300 µg, 100 µg -300 µg, 150 µg -300 µg, 200 µg -300 µg, 250 µg - 300 µg, 150 µg -200 µg, 150 µg -250 µg, 150 µg -300 µg, 200 µg -250 µg, or 250 µg -300 µg. For example, the effective amount may be a total dose of 25 µg, 50 µg, 55 µg, 60 µg, 65 µg, 70 µg, 75 µg, 80 µg, 85 µg, 90 µg, 95 µg, 100 µg, 110 µg, 120 µg, 130 µg, 140 µg, 150 µg, 160 µg, 170 µg, 180 µg, 190 µg, 200 µg, 210 µg, 220 µg, 230 µg, 240 µg, 250 µg, 260 µg, 270 µg, 280 µg, 290 µg, or 300 µg. In some embodiments, the effective amount is a total dose of 25 μg. In some embodiments, the effective amount is a total dose of 30 μg. In some embodiments, the effective amount is a total dose of 50 μg. In some embodiments, the effective amount is a total dose of 66 μg. In some embodiments, the effective amount is a total dose of 67 μg. In some embodiments, the effective amount is a total dose of 68 μg. In some embodiments, the effective
amount is a total dose of 132 μg. In some embodiments, the effective amount is a total dose of 133 μg. In some embodiments, the effective amount is a total dose of 134 μg. In some embodiments, the effective amount is a total dose of 266 μg. In some embodiments, the effective amount is a total dose of 267 μg. In some embodiments, the effective amount is a total dose of 268 μg. In some embodiments, the effective amount is a total dose of 100 μg. In some embodiments, the effective amount is a total dose of 200 μg. In some embodiments, the effective amount is a total dose of 300 μg. The RNA described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous). Vaccine Efficacy Some aspects of the present disclosure provide formulations of the immunizing compositions (e.g., RNA vaccines), wherein the RNA is formulated in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to an orthopoxvirus antigen). “An effective amount” is a dose of the RNA effective to produce an antigen-specific immune response. Also provided herein are methods of inducing an antigen- specific immune response in a subject. As used herein, an immune response to a vaccine or LNP of the present disclosure is the development in a subject of a humoral and/or a cellular immune response to a (one or more) orthopoxvirus protein(s) present in the vaccine. For purposes of the present disclosure, a “humoral” immune response refers to an immune response mediated by antibody molecules, including, e.g., secretory (IgA) or IgG molecules, while a “cellular” immune response is one mediated by T-lymphocytes (e.g., CD4+ helper and/or CD8+ T cells (e.g., CTLs) and/or other white blood cells. One important aspect of cellular immunity involves an antigen-specific response by cytolytic T-cells (CTLs). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves and antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function and focus the activity nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A cellular immune response also leads to the production of cytokines, chemokines, and other such molecules produced by activated T-cells and/or other white blood cells including those derived from CD4+ and CD8+ T-cells.
In some embodiments, the antigen-specific immune response is characterized by measuring an anti-orthopoxvirus antigen antibody titer produced in a subject administered an immunizing composition as provided herein. An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen or epitope of an antigen. Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example. In some embodiments, an antibody titer is used to assess whether a subject has had an infection or to determine whether immunizations are required. In some embodiments, an antibody titer is used to determine the strength of an autoimmune response, to determine whether a booster immunization is needed, to determine whether a previous vaccine was effective, and to identify any recent or prior infections. In accordance with the present disclosure, an antibody titer may be used to determine the strength of an immune response induced in a subject by an immunizing composition (e.g., RNA vaccine). In some embodiments, an anti-orthopoxvirus antigen antibody titer produced in a subject is increased by at least 1 log relative to a control. For example, anti-orthopoxvirus antigen antibody titer produced in a subject may be increased by at least 1.5, at least 2, at least 2.5, or at least 3 log relative to a control. In some embodiments, the anti-orthopoxvirus antigen antibody titer produced in the subject is increased by 1, 1.5, 2, 2.5 or 3 log relative to a control. In some embodiments, the anti-orthopoxvirus antigen antibody titer produced in the subject is increased by 1-3 log relative to a control. For example, the anti-orthopoxvirus virus antigen antibody titer produced in a subject may be increased by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to a control. In some embodiments, the anti-orthopoxvirus antigen antibody titer produced in a subject is increased at least 2 times relative to a control. For example, the anti-orthopoxvirus antigen antibody titer produced in a subject may be increased at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times relative to a control. In some embodiments, the anti-orthopoxvirus antigen antibody titer produced in the subject is increased 2, 3, 4, 5, 6, 7, 8, 9, or 10 times relative to a control. In some embodiments, the anti-orthopoxvirus antigen antibody titer produced in a subject is increased 2- 10 times relative to a control. For example, the anti-orthopoxvirus antigen antibody titer produced in a subject may be increased 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times relative to a control.
In some embodiments, an antigen-specific immune response is measured as a ratio of geometric mean titer (GMT), referred to as a geometric mean ratio (GMR), of serum neutralizing antibody titers to an orthopoxvirus. A geometric mean titer (GMT) is the average antibody titer for a group of subjects calculated by multiplying all values and taking the nth root of the number, where n is the number of subjects with available data. A control, in some embodiments, is an anti-orthopoxvirus antigen antibody titer produced in a subject who has not been administered an immunizing composition (e.g., RNA vaccine). In some embodiments, a control is an anti-orthopoxvirus antigen antibody titer produced in a subject administered a recombinant or purified protein vaccine. Recombinant protein vaccines typically include protein antigens that either have been produced in a heterologous expression system (e.g., bacteria or yeast) or purified from large amounts of the pathogenic organism. In some embodiments, the ability of an immunizing composition (e.g., RNA vaccine) to be effective is measured in a murine model. For example, an immunizing composition may be administered to a murine model and the murine model assayed for induction of neutralizing antibody titers. Viral challenge studies may also be used to assess the efficacy of a vaccine of the present disclosure. For example, an immunizing composition may be administered to a murine model, the murine model challenged with virus, and the murine model assayed for survival and/or immune response (e.g., neutralizing antibody response, T cell response (e.g., cytokine response)). A “standard of care,” as provided herein, refers to a medical or psychological treatment guideline and can be general or specific. “Standard of care” specifies appropriate treatment based on scientific evidence and collaboration between medical professionals involved in the treatment of a given condition. It is the diagnostic and treatment process that a physician/ clinician should follow for a certain type of patient, illness or clinical circumstance. A “standard of care dose,” as provided herein, refers to the dose of a recombinant or purified protein vaccine, or a live attenuated or inactivated vaccine, or a VLP vaccine, that a physician/clinician or other medical professional would administer to a subject to treat or prevent an orthopoxvirus infection or a related condition, while following the standard of care guideline for treating or preventing an orthopoxvirus infection or a related condition. In some embodiments, the anti-orthopoxvirus antigen antibody titer produced in a subject administered an effective amount of an immunizing composition is equivalent to an anti- orthopoxvirus antigen antibody titer produced in a control subject administered a standard of care dose of a recombinant or purified protein vaccine, or a live attenuated or inactivated vaccine, or a VLP vaccine.
Vaccine efficacy may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis.2010 Jun 1;201(11):1607-10). For example, vaccine efficacy may be measured by double-blind, randomized, clinical controlled trials. Vaccine efficacy may be expressed as a proportionate reduction in disease attack rate (AR) between the unvaccinated (ARU) and vaccinated (ARV) study cohorts and can be calculated from the relative risk (RR) of disease among the vaccinated group with use of the following formulas: Efficacy = (ARU – ARV)/ARU x 100; and Efficacy = (1-RR) x 100. Likewise, vaccine effectiveness may be assessed using standard analyses (see, e.g., Weinberg et al., J Infect Dis.2010 Jun 1;201(11):1607-10). Vaccine effectiveness is an assessment of how a vaccine (which may have already proven to have high vaccine efficacy) reduces disease in a population. This measure can assess the net balance of benefits and adverse effects of a vaccination program, not just the vaccine itself, under natural field conditions rather than in a controlled clinical trial. Vaccine effectiveness is proportional to vaccine efficacy (potency) but is also affected by how well target groups in the population are immunized, as well as by other non-vaccine-related factors that influence the ‘real-world’ outcomes of hospitalizations, ambulatory visits, or costs. For example, a retrospective case control analysis may be used, in which the rates of vaccination among a set of infected cases and appropriate controls are compared. Vaccine effectiveness may be expressed as a rate difference, with use of the odds ratio (OR) for developing infection despite vaccination: Effectiveness = (1 – OR) x 100. In some embodiments, efficacy of the immunizing composition (e.g., RNA vaccine) is at least 60% relative to unvaccinated control subjects. For example, efficacy of the immunizing composition may be at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 95%, at least 98%, or 100% relative to unvaccinated control subjects. Sterilizing Immunity. Sterilizing immunity refers to a unique immune status that prevents effective pathogen infection into the host. In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to provide sterilizing immunity in the subject for at least 1 year. For example, the effective amount of an immunizing composition of the present disclosure is sufficient to provide sterilizing immunity in the subject for at least 2 years, at least 3 years, at least 4 years, or at least 5 years. In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to provide sterilizing immunity in the subject at an at least 5-fold lower dose relative to control. For example, the effective amount may be sufficient to provide sterilizing immunity in the subject at an at least 10-fold lower, 15-fold, or 20-fold lower dose relative to a control.
Detectable Antigen. In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to produce detectable levels orthopoxvirus antigen as measured in serum of the subject at 1-72 hours post administration. Titer. An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen (e.g., an anti-orthopoxvirus antigen). Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example. In some embodiments, the effective amount of an immunizing composition of the present disclosure is sufficient to produce a 1,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the orthopoxvirus antigen as measured in serum of the subject at 1- 72 hours post administration. In some embodiments, the effective amount is sufficient to produce a 1,000-5,000 neutralizing antibody titer produced by neutralizing antibody against the orthopoxvirus antigen as measured in serum of the subject at 1-72 hours post administration. In some embodiments, the effective amount is sufficient to produce a 5,000-10,000 neutralizing antibody titer produced by neutralizing antibody against the orthopoxvirus antigen as measured in serum of the subject at 1-72 hours post administration. In some embodiments, the neutralizing antibody titer is at least 100 NT50. For example, the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NT50. In some embodiments, the neutralizing antibody titer is at least 10,000 NT50. In some embodiments, the neutralizing antibody titer is at least 100 neutralizing units per milliliter (NU/mL). For example, the neutralizing antibody titer may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000 NU/mL. In some embodiments, the neutralizing antibody titer is at least 10,000 NU/mL. In some embodiments, an anti-orthopoxvirus antigen antibody titer produced in the subject is increased by at least 1 log relative to a control. For example, an anti-orthopoxvirus antigen antibody titer produced in the subject may be increased by at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 log relative to a control. In some embodiments, an anti-orthopoxvirus antigen antibody titer produced in the subject is increased at least 2 times relative to a control. For example, an anti-orthopoxvirus antigen antibody titer produced in the subject is increased by at least 3, 4, 5, 6, 7, 8, 9 or 10 times relative to a control. In some embodiments, a geometric mean, which is the nth root of the product of n numbers, is generally used to describe proportional growth. Geometric mean, in some embodiments, is used to characterize antibody titer produced in a subject.
A control may be, for example, an unvaccinated subject, or a subject administered a live attenuated viral vaccine, an inactivated viral vaccine, or a protein subunit vaccine. As recently disclosed in Freyn, A. et al, A monkeypox mRNA-lipid nanoparticle vaccine targeting virus binding, entry, and transmission drives protection against lethal orthopoxviral challenge, bioRxiv 2022.12.17.520886, an mRNA-lipid nanoparticle vaccine encoding a set of four highly conserved monkeypox virus surface proteins involved in virus attachment, entry and transmission were demonstrated to induce MPXV-specific immunity and heterologous protection against a lethal vaccinia virus (VACV) challenge. The effect of the mRNA vaccine was compared to the current MPXV vaccine (Modified Vaccinia Virus Ankara (MVA)), and produced superior neutralizing and cellular spread-inhibitory activities against MPXV and VACV as well as greater Fc-effector Th1-biased humoral immunity to the four MPXV antigens and the four VACV homologs. Combinations of two, three or four MPXV antigen expressing mRNAs protected against disease-related weight loss and death. Multivalent MPXV mRNAs also resulted in superior cross-protection compared to MVA. The cross-protective response was associated with a combination of neutralizing and non-neutralizing antibody functions. Thus, an MPXV mRNA vaccine can induce robust neutralizing and functional cross-reactive antibodies able to confer comparable, if not superior, protection against a lethal challenge of an orthologous orthopoxvirus compared to MVA. SEQUENCE LISTING
Any of the mRNA sequences described herein may include a 5’ UTR and/or a 3’ UTR. The UTR sequences may be selected from any of the UTR sequences disclosed herein, or other known UTR sequences may be used. It should also be understood that any of the mRNAs described herein may further comprise a poly(A) tail and/or cap (e.g., 7mG(5’)ppp(5’)NlmpNp). Further, while many of the mRNAs and encoded antigen sequences described herein include a signal peptide and/or a peptide tag (e.g., C-terminal His tag), it should be understood that the indicated signal peptide and/or peptide tag may be substituted for a different signal peptide and/or peptide tag, or the signal peptide and/or peptide tag may be omitted. EQUIVALENTS All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document. The indefinite articles “a” and “an,” as used herein in the specification and in the embodiments and/or claims, unless clearly indicated to the contrary, should be understood to
mean “at least one.” It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. The terms “about” and “substantially” preceding a numerical value mean ±10% of the recited numerical value. Where a range of values is provided, each value between and including the upper and lower ends of the range are specifically contemplated and described herein.
Claims
CLAIMS 1. A composition, comprising: a first messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame (ORF) encoding a first orthopoxvirus protein and a lipid nanoparticle.
2. The composition of claim 1, further comprising a second mRNA polynucleotide comprising an ORF encoding a second orthopoxvirus protein.
3. The composition of claim 2, further comprising a third mRNA polynucleotide comprising an ORF encoding a third orthopoxvirus protein.
4. The composition of claim 3, further comprising a fourth mRNA polynucleotide comprising an ORF encoding a fourth orthopoxvirus protein.
5. The composition of any one of claims 1-4, wherein the first orthopoxvirus protein comprises a mature virus (MV) orthopoxvirus protein or an extracellular enveloped virus (EV) orthopoxvirus protein.
6. The composition of any one of claims 2-5, wherein the second orthopoxvirus protein comprises a MV orthopoxvirus protein or an EV orthopoxvirus protein.
7. The composition of any one of claims 3-6, wherein the third orthopoxvirus protein comprises an MV orthopoxvirus protein or an EV orthopoxvirus protein.
8. The composition of any one of claims 4-6, wherein the fourth orthopoxvirus protein comprises an MV orthopoxvirus protein or an EV orthopoxvirus protein.
9. The composition of any one of claims 5-8, wherein the MV orthopoxvirus protein is A29L or M1R.
10. The composition of any one of claims 5-9, wherein the EV orthopoxvirus protein is B6R or A35R.
11. The composition of any one of claims 4-10, wherein the first orthopoxvirus protein comprises A27L, the second orthopoxvirus protein comprises M1R, the third orthopoxvirus protein comprises B6R, and the fourth orthopoxvirus protein comprises A35R.
12. A composition comprising a first messenger ribonucleic acid (mRNA) polynucleotide comprising an open reading frame (ORF) encoding a first orthopoxvirus protein, and a lipid
nanoparticle, wherein the first orthopoxvirus protein is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 7-34.
13. The composition of claim 12, further comprising a second mRNA polynucleotide comprising an ORF encoding a second orthopoxvirus protein, wherein the second orthopoxvirus protein is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 7-34.
14. The composition of claim 13, further comprising a third mRNA polynucleotide comprising an ORF encoding a third orthopoxvirus protein, wherein the third orthopoxvirus protein is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 7-34.
15. The composition of claim 14, further comprising a fourth mRNA polynucleotide comprising an ORF encoding a fourth orthopoxvirus protein, wherein the fourth orthopoxvirus protein is at least 80% identical to any one of the amino acid sequences of SEQ ID NOs: 7-34 or an immunogenic fragment thereof.
16. The composition of any one of claims 12-15, wherein the orthopoxvirus protein is at least 90%, at least 95%, at least 97%, or at least 99% identical to any one of the amino acid sequences of SEQ ID NOs: 7-34.
17. The composition of claim 16, wherein the orthopoxvirus protein is identical to any one of the amino acid sequences of SEQ ID NOs: 7-34.
18. The composition of any one of claims 12-17, wherein the ORF comprises a sequence that is at least 90%, at least 95%, at least 97%, or at least 99% identical to any one of SEQ ID NOs: 35-62.
19. The composition of claim 18, wherein the ORF comprises a sequence that identical to any one of SEQ ID NOs: 35-62.
20. The composition of any one of claims 1-19, wherein the first, second, third, and/or fourth mRNA comprises a chemical modification.
21. The composition of claim 20, wherein the first, second, third, and/or fourth mRNA is fully modified.
22. The composition of claim 20 or 21, wherein the chemical modification is 1- methylpseudouridine.
23. The composition of any one of claims 1-22, wherein the lipid nanoparticle comprises a molar ratio of 20-60% ionizable amino lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5- 15% PEG-modified lipid.
24. The composition of claim 23, wherein the lipid nanoparticle comprises 1-5 mol% PEG- modified lipid; 10-20 mol% non-cationic lipid; 35-45 mol% sterol; and 40-50 mol% ionizable cationic lipid.
25. The composition of claim 23 or 24, wherein the PEG-modified lipid is 1,2 dimyristoyl- sn-glycerol, methoxypolyethyleneglycol (PEG2000 DMG), the non-cationic lipid is 1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC), the sterol is cholesterol; and the ionizable cationic lipid has the structure of Compound 1:
(Compound 1).
26. A method for vaccinating a subject, comprising: administering to the subject the composition of any one of claims 1-25.
27. The method of claim 26, wherein the method prevents an orthopoxvirus infection in the subject.
28. The method of claim 26, wherein the method reduces the severity of an orthopoxvirus infection in the subject.
29. The method of claim 26, wherein the subject is seronegative for an orthopoxvirus.
30. The method of claim 26, wherein the subject is seropositive for an orthopoxvirus.
31. A multivalent RNA composition, comprising: at least two messenger ribonucleic acid (mRNA) polynucleotides, each comprising an open reading frame (ORF) encoding a monkeypox antigen and a lipid nanoparticle.
32. The composition of claim 31, wherein the monkeypox antigen comprises a mature virus (MV) orthopoxvirus protein and/or an extracellular enveloped virus (EV) orthopoxvirus protein.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202263345390P | 2022-05-24 | 2022-05-24 | |
US63/345,390 | 2022-05-24 | ||
US202263395567P | 2022-08-05 | 2022-08-05 | |
US63/395,567 | 2022-08-05 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2023230481A1 true WO2023230481A1 (en) | 2023-11-30 |
WO2023230481A8 WO2023230481A8 (en) | 2024-01-25 |
Family
ID=86776294
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2023/067368 WO2023230481A1 (en) | 2022-05-24 | 2023-05-23 | Orthopoxvirus vaccines |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2023230481A1 (en) |
Citations (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002098443A2 (en) | 2001-06-05 | 2002-12-12 | Curevac Gmbh | Stabilised mrna with an increased g/c content and optimised codon for use in gene therapy |
US20090226470A1 (en) | 2007-12-11 | 2009-09-10 | Mauro Vincent P | Compositions and methods related to mRNA translational enhancer elements |
US20100129877A1 (en) | 2005-09-28 | 2010-05-27 | Ugur Sahin | Modification of RNA, Producing an Increased Transcript Stability and Translation Efficiency |
US20100293625A1 (en) | 2007-09-26 | 2010-11-18 | Interexon Corporation | Synthetic 5'UTRs, Expression Vectors, and Methods for Increasing Transgene Expression |
US8278063B2 (en) | 2007-06-29 | 2012-10-02 | Commonwealth Scientific And Industrial Research Organisation | Methods for degrading toxic compounds |
WO2013185069A1 (en) | 2012-06-08 | 2013-12-12 | Shire Human Genetic Therapies, Inc. | Pulmonary delivery of mrna to non-lung target cells |
US20140206753A1 (en) | 2011-06-08 | 2014-07-24 | Shire Human Genetic Therapies, Inc. | Lipid nanoparticle compositions and methods for mrna delivery |
WO2014144196A1 (en) | 2013-03-15 | 2014-09-18 | Shire Human Genetic Therapies, Inc. | Synergistic enhancement of the delivery of nucleic acids via blended formulations |
WO2015024667A1 (en) | 2013-08-21 | 2015-02-26 | Curevac Gmbh | Method for increasing expression of rna-encoded proteins |
US9012219B2 (en) | 2005-08-23 | 2015-04-21 | The Trustees Of The University Of Pennsylvania | RNA preparations comprising purified modified RNA for reprogramming cells |
WO2015062738A1 (en) | 2013-11-01 | 2015-05-07 | Curevac Gmbh | Modified rna with decreased immunostimulatory properties |
WO2015101414A2 (en) | 2013-12-30 | 2015-07-09 | Curevac Gmbh | Artificial nucleic acid molecules |
WO2015101415A1 (en) | 2013-12-30 | 2015-07-09 | Curevac Gmbh | Artificial nucleic acid molecules |
WO2017127750A1 (en) | 2016-01-22 | 2017-07-27 | Modernatx, Inc. | Messenger ribonucleic acids for the production of intracellular binding polypeptides and methods of use thereof |
WO2019036682A1 (en) | 2017-08-18 | 2019-02-21 | Modernatx, Inc. | Rna polymerase variants |
WO2020172239A1 (en) | 2019-02-20 | 2020-08-27 | Modernatx, Inc. | Rna polymerase variants for co-transcriptional capping |
-
2023
- 2023-05-23 WO PCT/US2023/067368 patent/WO2023230481A1/en unknown
Patent Citations (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002098443A2 (en) | 2001-06-05 | 2002-12-12 | Curevac Gmbh | Stabilised mrna with an increased g/c content and optimised codon for use in gene therapy |
US9012219B2 (en) | 2005-08-23 | 2015-04-21 | The Trustees Of The University Of Pennsylvania | RNA preparations comprising purified modified RNA for reprogramming cells |
US20100129877A1 (en) | 2005-09-28 | 2010-05-27 | Ugur Sahin | Modification of RNA, Producing an Increased Transcript Stability and Translation Efficiency |
US8278063B2 (en) | 2007-06-29 | 2012-10-02 | Commonwealth Scientific And Industrial Research Organisation | Methods for degrading toxic compounds |
US20100293625A1 (en) | 2007-09-26 | 2010-11-18 | Interexon Corporation | Synthetic 5'UTRs, Expression Vectors, and Methods for Increasing Transgene Expression |
US20090226470A1 (en) | 2007-12-11 | 2009-09-10 | Mauro Vincent P | Compositions and methods related to mRNA translational enhancer elements |
US20140206753A1 (en) | 2011-06-08 | 2014-07-24 | Shire Human Genetic Therapies, Inc. | Lipid nanoparticle compositions and methods for mrna delivery |
WO2013185069A1 (en) | 2012-06-08 | 2013-12-12 | Shire Human Genetic Therapies, Inc. | Pulmonary delivery of mrna to non-lung target cells |
WO2014144196A1 (en) | 2013-03-15 | 2014-09-18 | Shire Human Genetic Therapies, Inc. | Synergistic enhancement of the delivery of nucleic acids via blended formulations |
WO2015024667A1 (en) | 2013-08-21 | 2015-02-26 | Curevac Gmbh | Method for increasing expression of rna-encoded proteins |
WO2015062738A1 (en) | 2013-11-01 | 2015-05-07 | Curevac Gmbh | Modified rna with decreased immunostimulatory properties |
WO2015101414A2 (en) | 2013-12-30 | 2015-07-09 | Curevac Gmbh | Artificial nucleic acid molecules |
WO2015101415A1 (en) | 2013-12-30 | 2015-07-09 | Curevac Gmbh | Artificial nucleic acid molecules |
WO2017127750A1 (en) | 2016-01-22 | 2017-07-27 | Modernatx, Inc. | Messenger ribonucleic acids for the production of intracellular binding polypeptides and methods of use thereof |
WO2019036682A1 (en) | 2017-08-18 | 2019-02-21 | Modernatx, Inc. | Rna polymerase variants |
WO2020172239A1 (en) | 2019-02-20 | 2020-08-27 | Modernatx, Inc. | Rna polymerase variants for co-transcriptional capping |
Non-Patent Citations (21)
Title |
---|
"Remington: The Science and Practice of Pharmacy", 2005, LIPPINCOTT WILLIAMS & WILKINS |
CHO K.J. ET AL., J MOL BIOL., vol. 390, 2009, pages 83 - 98 |
CORBETT KIZZMEKIA S. ET AL: "Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates", THE NEW ENGLAND JOURNAL OF MEDICINE, vol. 383, no. 16, 15 October 2020 (2020-10-15), US, pages 1544 - 1555, XP055949061, ISSN: 0028-4793, Retrieved from the Internet <URL:https://www.nejm.org/doi/pdf/10.1056/NEJMoa2024671?articleTools=true> DOI: 10.1056/NEJMoa2024671 * |
FREYN ALEC W. ET AL: "A monkeypox mRNA-lipid nanoparticle vaccine targeting virus binding, entry, and transmission drives protection against lethal orthopoxviral challenge", BIORXIV, 19 December 2022 (2022-12-19), XP093082340, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2022.12.17.520886v1.full.pdf> [retrieved on 20230914], DOI: 10.1101/2022.12.17.520886 * |
FREYN, A.: "A monkeypox mRNA-lipid nanoparticle vaccine targeting virus binding, entry, and transmission drives protection against lethal orthopoxviral challenge", BIORXIV 2022.12.17.520886 |
GRANIER T. ET AL., JBIOL INORG CHEM., vol. 8, 2003, pages 105 - 111 |
HERAUD JEAN-MICHEL ET AL: "Subunit Recombinant Vaccine Protects against Monkeypox", THE JOURNAL OF IMMUNOLOGY, vol. 177, no. 4, 15 August 2006 (2006-08-15), US, pages 2552 - 2564, XP093082339, ISSN: 0022-1767, Retrieved from the Internet <URL:https://watermark.silverchair.com/zim01606002552.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAABcIwggW-BgkqhkiG9w0BBwagggWvMIIFqwIBADCCBaQGCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMpvMBNZRvUFQ5J6UhAgEQgIIFdeGmUNtrn2POQBnULMjdG8NqRAW1e0kAPj8Nrlh6DBfmdxhSiwmQA7nWTY7iBDtNMdVVZakknawcnkYMMP-5z> DOI: 10.4049/jimmunol.177.4.2552 * |
KIM, J.H. ET AL., PLOS ONE, vol. 6, 2011, pages e18556 |
LAWSON D.M. ET AL., NATURE, vol. 349, 1991, pages 541 - 544 |
LOPEZ-SAGASETA, J. ET AL., COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL, vol. 14, 2016, pages 58 - 68 |
NEEDLEMAN, S.B.WUNSCH, C.D.: "A general method applicable to the search for similarities in the amino acid sequences of two proteins.", J. MOL. BIOL., vol. 48, 1970, pages 443 - 453 |
RAHMANPOUR R. ET AL., FEBS J., vol. 280, 2013, pages 2097 - 2104 |
SAMBROOK: "Joseph. Molecular Cloning : a Laboratory Manual.", 2001, COLD SPRING HARBOR LABORATORY PRESS |
SANG YE ET AL: "Monkeypox virus quadrivalent mRNA vaccine induces immune response and protects against vaccinia virus", SIGNAL TRANSDUCTION AND TARGETED THERAPY, vol. 8, no. 1, 28 April 2023 (2023-04-28), XP093082342, Retrieved from the Internet <URL:https://www.nature.com/articles/s41392-023-01432-5> DOI: 10.1038/s41392-023-01432-5 * |
SMITH, T.F.WATERMAN, M.S.: "Identification of common molecular subsequences.", J. MOL. BIOL., vol. 147, 1981, pages 195 - 197, XP024015032, DOI: 10.1016/0022-2836(81)90087-5 |
STEPHEN F. ALTSCHUL ET AL.: "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402, XP002905950, DOI: 10.1093/nar/25.17.3389 |
SUTTER M. ET AL., NAT STRUCT MOL BIOL., vol. 15, 2008, pages 939 - 947 |
TAO Y ET AL., STRUCTURE, vol. 5, no. 6, 15 June 1997 (1997-06-15), pages 789 - 98 |
WEINBERG ET AL., J INFECT DIS., vol. 201, no. 11, 1 June 2010 (2010-06-01), pages 1607 - 10 |
ZHANG RONG-RONG ET AL: "Rational development of multicomponent mRNA vaccine candidates against mpox", EMERGING MICROBES & INFECTIONS, vol. 12, no. 1, 31 March 2023 (2023-03-31), XP093082341, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10071941/pdf/TEMI_12_2192815.pdf> DOI: 10.1080/22221751.2023.2192815 * |
ZHANG X. ET AL., J MOL BIOL., vol. 362, 2006, pages 753 - 770 |
Also Published As
Publication number | Publication date |
---|---|
WO2023230481A8 (en) | 2024-01-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20210228707A1 (en) | Coronavirus rna vaccines | |
US20230108894A1 (en) | Coronavirus rna vaccines | |
US20230346914A1 (en) | Sars-cov-2 mrna domain vaccines | |
US20230355743A1 (en) | Multi-proline-substituted coronavirus spike protein vaccines | |
US11351242B1 (en) | HMPV/hPIV3 mRNA vaccine composition | |
WO2021211343A1 (en) | Zika virus mrna vaccines | |
WO2021222304A1 (en) | Sars-cov-2 rna vaccines | |
WO2019148101A1 (en) | Rsv rna vaccines | |
EP4277653A1 (en) | Variant strain-based coronavirus vaccines | |
WO2022221440A1 (en) | Influenza-coronavirus combination vaccines | |
WO2022221359A1 (en) | Epstein-barr virus mrna vaccines | |
WO2022221335A9 (en) | Respiratory virus combination vaccines | |
EP4355891A1 (en) | Coronavirus glycosylation variant vaccines | |
WO2022221336A1 (en) | Respiratory syncytial virus mrna vaccines | |
WO2023283642A2 (en) | Pan-human coronavirus concatemeric vaccines | |
WO2023283651A1 (en) | Pan-human coronavirus vaccines | |
EP4355761A1 (en) | Mrna vaccines encoding flexible coronavirus spike proteins | |
WO2023283645A1 (en) | Pan-human coronavirus domain vaccines | |
WO2023092069A1 (en) | Sars-cov-2 mrna domain vaccines and methods of use | |
WO2023230481A1 (en) | Orthopoxvirus vaccines | |
US20240139309A1 (en) | Variant strain-based coronavirus vaccines |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23731487 Country of ref document: EP Kind code of ref document: A1 |