WO2023211281A1 - Antiviral vaccine composition - Google Patents
Antiviral vaccine composition Download PDFInfo
- Publication number
- WO2023211281A1 WO2023211281A1 PCT/NL2023/050232 NL2023050232W WO2023211281A1 WO 2023211281 A1 WO2023211281 A1 WO 2023211281A1 NL 2023050232 W NL2023050232 W NL 2023050232W WO 2023211281 A1 WO2023211281 A1 WO 2023211281A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cpg
- protein
- sars
- vaccine composition
- cov
- Prior art date
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 61
- 229960004854 viral vaccine Drugs 0.000 title claims abstract description 38
- 239000002671 adjuvant Substances 0.000 claims abstract description 92
- 102000036639 antigens Human genes 0.000 claims abstract description 45
- 108091007433 antigens Proteins 0.000 claims abstract description 45
- 239000000427 antigen Substances 0.000 claims abstract description 43
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 39
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 38
- 229940044665 STING agonist Drugs 0.000 claims abstract description 34
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 32
- 108090000565 Capsid Proteins Proteins 0.000 claims abstract description 9
- 210000000234 capsid Anatomy 0.000 claims abstract description 9
- 239000011159 matrix material Substances 0.000 claims abstract description 9
- 108090001074 Nucleocapsid Proteins Proteins 0.000 claims description 36
- 108091006197 SARS-CoV-2 Nucleocapsid Protein Proteins 0.000 claims description 33
- 241000700605 Viruses Species 0.000 claims description 23
- 230000003612 virological effect Effects 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 18
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 claims description 12
- 241000712461 unidentified influenza virus Species 0.000 claims description 9
- 241000725643 Respiratory syncytial virus Species 0.000 claims description 8
- 208000036142 Viral infection Diseases 0.000 claims description 8
- 208000015181 infectious disease Diseases 0.000 claims description 8
- 230000009385 viral infection Effects 0.000 claims description 8
- 108010052285 Membrane Proteins Proteins 0.000 claims description 7
- 238000011282 treatment Methods 0.000 claims description 7
- 102000018697 Membrane Proteins Human genes 0.000 claims description 6
- 239000012646 vaccine adjuvant Substances 0.000 claims description 6
- 241000711573 Coronaviridae Species 0.000 claims description 5
- 241000315672 SARS coronavirus Species 0.000 claims description 5
- 108010067390 Viral Proteins Proteins 0.000 claims description 5
- PDXMFTWFFKBFIN-XPWFQUROSA-N cyclic di-AMP Chemical group C([C@H]1O2)OP(O)(=O)O[C@H]3[C@@H](O)[C@H](N4C5=NC=NC(N)=C5N=C4)O[C@@H]3COP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 PDXMFTWFFKBFIN-XPWFQUROSA-N 0.000 claims description 4
- 230000002265 prevention Effects 0.000 claims description 4
- 230000001932 seasonal effect Effects 0.000 claims description 4
- 208000025721 COVID-19 Diseases 0.000 claims description 3
- 208000037847 SARS-CoV-2-infection Diseases 0.000 claims description 3
- 239000003981 vehicle Substances 0.000 claims description 3
- 241000709661 Enterovirus Species 0.000 claims description 2
- 241000712431 Influenza A virus Species 0.000 claims description 2
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 claims description 2
- 241000351643 Metapneumovirus Species 0.000 claims description 2
- 241001263478 Norovirus Species 0.000 claims description 2
- 208000002606 Paramyxoviridae Infections Diseases 0.000 claims description 2
- 239000008135 aqueous vehicle Substances 0.000 claims description 2
- 208000037797 influenza A Diseases 0.000 claims description 2
- 229960005486 vaccine Drugs 0.000 description 61
- 108090000765 processed proteins & peptides Proteins 0.000 description 52
- 229940046168 CpG oligodeoxynucleotide Drugs 0.000 description 48
- 210000004027 cell Anatomy 0.000 description 42
- 239000003814 drug Substances 0.000 description 29
- 102000004196 processed proteins & peptides Human genes 0.000 description 28
- 210000001744 T-lymphocyte Anatomy 0.000 description 26
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 24
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 23
- 239000011780 sodium chloride Substances 0.000 description 20
- 238000002255 vaccination Methods 0.000 description 20
- 239000003795 chemical substances by application Substances 0.000 description 19
- 241000699670 Mus sp. Species 0.000 description 18
- 101001024637 Severe acute respiratory syndrome coronavirus 2 Nucleoprotein Proteins 0.000 description 18
- 230000036039 immunity Effects 0.000 description 18
- 239000007924 injection Substances 0.000 description 18
- 238000002347 injection Methods 0.000 description 18
- 239000002773 nucleotide Substances 0.000 description 17
- 125000003729 nucleotide group Chemical group 0.000 description 17
- 230000003389 potentiating effect Effects 0.000 description 17
- 241001465754 Metazoa Species 0.000 description 16
- 206010028980 Neoplasm Diseases 0.000 description 16
- 201000011510 cancer Diseases 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 230000003053 immunization Effects 0.000 description 13
- 238000002649 immunization Methods 0.000 description 13
- 238000004519 manufacturing process Methods 0.000 description 13
- 150000003839 salts Chemical class 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 230000028993 immune response Effects 0.000 description 12
- 230000001939 inductive effect Effects 0.000 description 12
- 230000004044 response Effects 0.000 description 12
- 229940124597 therapeutic agent Drugs 0.000 description 12
- 241001678559 COVID-19 virus Species 0.000 description 11
- 108010002616 Interleukin-5 Proteins 0.000 description 11
- 102000000743 Interleukin-5 Human genes 0.000 description 11
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 102000004127 Cytokines Human genes 0.000 description 10
- 108090000695 Cytokines Proteins 0.000 description 10
- 210000003719 b-lymphocyte Anatomy 0.000 description 10
- 230000007969 cellular immunity Effects 0.000 description 10
- 210000004544 dc2 Anatomy 0.000 description 10
- 239000002245 particle Substances 0.000 description 10
- 210000004988 splenocyte Anatomy 0.000 description 10
- 238000010186 staining Methods 0.000 description 10
- 102100037850 Interferon gamma Human genes 0.000 description 9
- 108010074328 Interferon-gamma Proteins 0.000 description 9
- 101710141454 Nucleoprotein Proteins 0.000 description 9
- 230000005867 T cell response Effects 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 9
- 239000004615 ingredient Substances 0.000 description 9
- 239000002105 nanoparticle Substances 0.000 description 9
- 238000003114 enzyme-linked immunosorbent spot assay Methods 0.000 description 8
- 230000003834 intracellular effect Effects 0.000 description 8
- 238000011510 Elispot assay Methods 0.000 description 7
- 230000014102 antigen processing and presentation of exogenous peptide antigen via MHC class I Effects 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 230000000069 prophylactic effect Effects 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000011740 C57BL/6 mouse Methods 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 241000282412 Homo Species 0.000 description 6
- 206010020751 Hypersensitivity Diseases 0.000 description 6
- 230000007815 allergy Effects 0.000 description 6
- 230000000840 anti-viral effect Effects 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 125000004122 cyclic group Chemical group 0.000 description 6
- 230000003308 immunostimulating effect Effects 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 244000052769 pathogen Species 0.000 description 6
- 229920000136 polysorbate Polymers 0.000 description 6
- YYGNTYWPHWGJRM-AAJYLUCBSA-N squalene Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C=C(/C)CC\C=C(/C)CCC=C(C)C YYGNTYWPHWGJRM-AAJYLUCBSA-N 0.000 description 6
- 238000002560 therapeutic procedure Methods 0.000 description 6
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 5
- 108090001005 Interleukin-6 Proteins 0.000 description 5
- 229930040373 Paraformaldehyde Natural products 0.000 description 5
- 239000000556 agonist Substances 0.000 description 5
- 208000026935 allergic disease Diseases 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 230000005847 immunogenicity Effects 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 210000001165 lymph node Anatomy 0.000 description 5
- 230000001404 mediated effect Effects 0.000 description 5
- 229920002866 paraformaldehyde Polymers 0.000 description 5
- 238000012552 review Methods 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 108090000672 Annexin A5 Proteins 0.000 description 4
- 102000004121 Annexin A5 Human genes 0.000 description 4
- 108010047761 Interferon-alpha Proteins 0.000 description 4
- 102000006992 Interferon-alpha Human genes 0.000 description 4
- 101500027983 Rattus norvegicus Octadecaneuropeptide Proteins 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 102000008235 Toll-Like Receptor 9 Human genes 0.000 description 4
- 108010060818 Toll-Like Receptor 9 Proteins 0.000 description 4
- 229940037003 alum Drugs 0.000 description 4
- 230000021615 conjugation Effects 0.000 description 4
- 230000016396 cytokine production Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 230000011488 interferon-alpha production Effects 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 239000002502 liposome Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 239000008194 pharmaceutical composition Substances 0.000 description 4
- 239000000546 pharmaceutical excipient Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 230000004936 stimulating effect Effects 0.000 description 4
- 230000032258 transport Effects 0.000 description 4
- 230000014567 type I interferon production Effects 0.000 description 4
- 229940124931 vaccine adjuvant Drugs 0.000 description 4
- 239000011534 wash buffer Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 description 3
- XRILCFTWUCUKJR-INFSMZHSSA-N 2'-3'-cGAMP Chemical compound C([C@H]([C@H]1O)O2)OP(O)(=O)O[C@H]3[C@@H](O)[C@H](N4C5=NC=NC(N)=C5N=C4)O[C@@H]3COP(O)(=O)O[C@H]1[C@@H]2N1C=NC2=C1NC(N)=NC2=O XRILCFTWUCUKJR-INFSMZHSSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 230000003844 B-cell-activation Effects 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 102000014914 Carrier Proteins Human genes 0.000 description 3
- 108010078791 Carrier Proteins Proteins 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 239000012981 Hank's balanced salt solution Substances 0.000 description 3
- 102000003945 NF-kappa B Human genes 0.000 description 3
- 108010057466 NF-kappa B Proteins 0.000 description 3
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000024932 T cell mediated immunity Effects 0.000 description 3
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 description 3
- 210000000447 Th1 cell Anatomy 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 239000013566 allergen Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000000612 antigen-presenting cell Anatomy 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 208000006673 asthma Diseases 0.000 description 3
- RFCBNSCSPXMEBK-INFSMZHSSA-N c-GMP-AMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]3[C@@H](O)[C@H](N4C5=NC=NC(N)=C5N=C4)O[C@@H]3COP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 RFCBNSCSPXMEBK-INFSMZHSSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical class NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 3
- 210000004443 dendritic cell Anatomy 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 3
- 239000002552 dosage form Substances 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000011194 good manufacturing practice Methods 0.000 description 3
- 230000004727 humoral immunity Effects 0.000 description 3
- 239000002955 immunomodulating agent Substances 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 150000002632 lipids Chemical class 0.000 description 3
- 108700021021 mRNA Vaccine Proteins 0.000 description 3
- 229940126582 mRNA vaccine Drugs 0.000 description 3
- 229940035032 monophosphoryl lipid a Drugs 0.000 description 3
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 3
- 230000003472 neutralizing effect Effects 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 239000012453 solvate Substances 0.000 description 3
- 229940031439 squalene Drugs 0.000 description 3
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 230000004083 survival effect Effects 0.000 description 3
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 2
- 208000035285 Allergic Seasonal Rhinitis Diseases 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 102100031256 Cyclic GMP-AMP synthase Human genes 0.000 description 2
- 108030002637 Cyclic GMP-AMP synthases Proteins 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- -1 DMXAA Chemical compound 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 101000643024 Homo sapiens Stimulator of interferon genes protein Proteins 0.000 description 2
- 101000800479 Homo sapiens Toll-like receptor 9 Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 102000002227 Interferon Type I Human genes 0.000 description 2
- 108010014726 Interferon Type I Proteins 0.000 description 2
- 102000014150 Interferons Human genes 0.000 description 2
- 108010050904 Interferons Proteins 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 108010058846 Ovalbumin Proteins 0.000 description 2
- 108010089430 Phosphoproteins Proteins 0.000 description 2
- 102000007982 Phosphoproteins Human genes 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 102100035533 Stimulator of interferon genes protein Human genes 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 102000002689 Toll-like receptor Human genes 0.000 description 2
- 108020000411 Toll-like receptor Proteins 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 239000003708 ampul Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 235000010980 cellulose Nutrition 0.000 description 2
- 210000000172 cytosol Anatomy 0.000 description 2
- 231100000433 cytotoxic Toxicity 0.000 description 2
- 230000001472 cytotoxic effect Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 2
- 238000007876 drug discovery Methods 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 102000045710 human TLR9 Human genes 0.000 description 2
- 230000028996 humoral immune response Effects 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 230000000091 immunopotentiator Effects 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 238000000099 in vitro assay Methods 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 206010022000 influenza Diseases 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- PGHMRUGBZOYCAA-ADZNBVRBSA-N ionomycin Chemical compound O1[C@H](C[C@H](O)[C@H](C)[C@H](O)[C@H](C)/C=C/C[C@@H](C)C[C@@H](C)C(/O)=C/C(=O)[C@@H](C)C[C@@H](C)C[C@@H](CCC(O)=O)C)CC[C@@]1(C)[C@@H]1O[C@](C)([C@@H](C)O)CC1 PGHMRUGBZOYCAA-ADZNBVRBSA-N 0.000 description 2
- PGHMRUGBZOYCAA-UHFFFAOYSA-N ionomycin Natural products O1C(CC(O)C(C)C(O)C(C)C=CCC(C)CC(C)C(O)=CC(=O)C(C)CC(C)CC(CCC(O)=O)C)CCC1(C)C1OC(C)(C(C)O)CC1 PGHMRUGBZOYCAA-UHFFFAOYSA-N 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 239000007788 liquid Substances 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
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000003094 microcapsule Substances 0.000 description 2
- 239000011859 microparticle Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- 230000000242 pagocytic effect Effects 0.000 description 2
- 229940023041 peptide vaccine Drugs 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 150000004713 phosphodiesters Chemical class 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 229960004793 sucrose Drugs 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 229940044655 toll-like receptor 9 agonist Drugs 0.000 description 2
- 238000011830 transgenic mouse model Methods 0.000 description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- 230000029069 type 2 immune response Effects 0.000 description 2
- 229950008737 vadimezan Drugs 0.000 description 2
- XGOYIMQSIKSOBS-UHFFFAOYSA-N vadimezan Chemical compound C1=CC=C2C(=O)C3=CC=C(C)C(C)=C3OC2=C1CC(O)=O XGOYIMQSIKSOBS-UHFFFAOYSA-N 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- JXTRMJLMBNFXEW-JZBGLOBFSA-N (2r,3r,4s,5r)-2-(6-aminopurin-9-yl)-5-(hydroxymethyl)oxolane-3,4-diol;phosphoric acid Chemical compound OP(O)(O)=O.C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O.C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O JXTRMJLMBNFXEW-JZBGLOBFSA-N 0.000 description 1
- JVJGCCBAOOWGEO-RUTPOYCXSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-4-amino-2-[[(2s,3s)-2-[[(2s,3s)-2-[[(2s)-2-azaniumyl-3-hydroxypropanoyl]amino]-3-methylpentanoyl]amino]-3-methylpentanoyl]amino]-4-oxobutanoyl]amino]-3-phenylpropanoyl]amino]-4-carboxylatobutanoyl]amino]-6-azaniumy Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O)CC1=CC=CC=C1 JVJGCCBAOOWGEO-RUTPOYCXSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- MIJDSYMOBYNHOT-UHFFFAOYSA-N 2-(ethylamino)ethanol Chemical compound CCNCCO MIJDSYMOBYNHOT-UHFFFAOYSA-N 0.000 description 1
- FAFONCPHZLORMH-INFSMZHSSA-N 3'2'-cgamp Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H]1OP(O)(=O)OC1)O)N2C=NC=3C(=O)N=C(NC=32)N)OP(O)(=O)O[C@H]2[C@H](N3C4=NC=NC(N)=C4N=C3)O[C@H]1[C@H]2O FAFONCPHZLORMH-INFSMZHSSA-N 0.000 description 1
- 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 1
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 1
- 102100039819 Actin, alpha cardiac muscle 1 Human genes 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- 206010010744 Conjunctivitis allergic Diseases 0.000 description 1
- 241000494545 Cordyline virus 2 Species 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 108700002099 Coronavirus Nucleocapsid Proteins Proteins 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
- 206010013700 Drug hypersensitivity Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 208000004262 Food Hypersensitivity Diseases 0.000 description 1
- 206010016946 Food allergy Diseases 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical class 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 1
- 101710154606 Hemagglutinin Proteins 0.000 description 1
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 1
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 1
- 101000959247 Homo sapiens Actin, alpha cardiac muscle 1 Proteins 0.000 description 1
- 101001032342 Homo sapiens Interferon regulatory factor 7 Proteins 0.000 description 1
- 101000917826 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-a Proteins 0.000 description 1
- 101000917824 Homo sapiens Low affinity immunoglobulin gamma Fc region receptor II-b Proteins 0.000 description 1
- 101000665442 Homo sapiens Serine/threonine-protein kinase TBK1 Proteins 0.000 description 1
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100038070 Interferon regulatory factor 7 Human genes 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- NNJVILVZKWQKPM-UHFFFAOYSA-N Lidocaine Chemical compound CCN(CC)CC(=O)NC1=C(C)C=CC=C1C NNJVILVZKWQKPM-UHFFFAOYSA-N 0.000 description 1
- 102100029204 Low affinity immunoglobulin gamma Fc region receptor II-a Human genes 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 1
- 108010006232 Neuraminidase Proteins 0.000 description 1
- 102000005348 Neuraminidase Human genes 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 102000011931 Nucleoproteins Human genes 0.000 description 1
- 108010061100 Nucleoproteins Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 1
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 1
- 108091081548 Palindromic sequence Proteins 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 101710176177 Protein A56 Proteins 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 206010039085 Rhinitis allergic Diseases 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 102100038192 Serine/threonine-protein kinase TBK1 Human genes 0.000 description 1
- 101001024647 Severe acute respiratory syndrome coronavirus Nucleoprotein Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- FEWJPZIEWOKRBE-UHFFFAOYSA-N Tartaric acid Natural products [H+].[H+].[O-]C(=O)C(O)C(O)C([O-])=O FEWJPZIEWOKRBE-UHFFFAOYSA-N 0.000 description 1
- 210000004241 Th2 cell Anatomy 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 208000024780 Urticaria Diseases 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- DHKHKXVYLBGOIT-UHFFFAOYSA-N acetaldehyde Diethyl Acetal Natural products CCOC(C)OCC DHKHKXVYLBGOIT-UHFFFAOYSA-N 0.000 description 1
- 150000001241 acetals Chemical class 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- VJHCJDRQFCCTHL-UHFFFAOYSA-N acetic acid 2,3,4,5,6-pentahydroxyhexanal Chemical compound CC(O)=O.OCC(O)C(O)C(O)C(O)C=O VJHCJDRQFCCTHL-UHFFFAOYSA-N 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 229940021704 adenovirus vaccine Drugs 0.000 description 1
- 238000012382 advanced drug delivery Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000001270 agonistic effect Effects 0.000 description 1
- 229940074608 allergen extract Drugs 0.000 description 1
- 229960004784 allergens Drugs 0.000 description 1
- 208000002205 allergic conjunctivitis Diseases 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 201000010105 allergic rhinitis Diseases 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- AZDRQVAHHNSJOQ-UHFFFAOYSA-N alumane Chemical class [AlH3] AZDRQVAHHNSJOQ-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 159000000013 aluminium salts Chemical class 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 239000002269 analeptic agent Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 230000005809 anti-tumor immunity Effects 0.000 description 1
- 229940121357 antivirals Drugs 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 239000012062 aqueous buffer Substances 0.000 description 1
- 208000024998 atopic conjunctivitis Diseases 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- LFYJSSARVMHQJB-QIXNEVBVSA-N bakuchiol Chemical compound CC(C)=CCC[C@@](C)(C=C)\C=C\C1=CC=C(O)C=C1 LFYJSSARVMHQJB-QIXNEVBVSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- PKFDLKSEZWEFGL-MHARETSRSA-N c-di-GMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]3[C@@H](O)[C@H](N4C5=C(C(NC(N)=N5)=O)N=C4)O[C@@H]3COP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 PKFDLKSEZWEFGL-MHARETSRSA-N 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 229950008138 carmellose Drugs 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 208000029771 childhood onset asthma Diseases 0.000 description 1
- 235000020971 citrus fruits Nutrition 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 229940028617 conventional vaccine Drugs 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- CTMZLDSMFCVUNX-VMIOUTBZSA-N cytidylyl-(3'->5')-guanosine Chemical group O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=C(C(N=C(N)N3)=O)N=C2)O)[C@@H](CO)O1 CTMZLDSMFCVUNX-VMIOUTBZSA-N 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 239000003405 delayed action preparation Substances 0.000 description 1
- 238000000586 desensitisation Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 235000013681 dietary sucrose Nutrition 0.000 description 1
- CRVGKGJPQYZRPT-UHFFFAOYSA-N diethylamino acetate Chemical compound CCN(CC)OC(C)=O CRVGKGJPQYZRPT-UHFFFAOYSA-N 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 201000005311 drug allergy Diseases 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 230000001815 facial effect Effects 0.000 description 1
- 229960004887 ferric hydroxide Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000020932 food allergy Nutrition 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- 229940075507 glyceryl monostearate Drugs 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000000185 hemagglutinin Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 235000011167 hydrochloric acid Nutrition 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 1
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 1
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 1
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 208000030603 inherited susceptibility to asthma Diseases 0.000 description 1
- 229940102223 injectable solution Drugs 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 230000014828 interferon-gamma production Effects 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 230000017306 interleukin-6 production Effects 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 238000010212 intracellular staining Methods 0.000 description 1
- IEECXTSVVFWGSE-UHFFFAOYSA-M iron(3+);oxygen(2-);hydroxide Chemical compound [OH-].[O-2].[Fe+3] IEECXTSVVFWGSE-UHFFFAOYSA-M 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000021633 leukocyte mediated immunity Effects 0.000 description 1
- 229960004194 lidocaine Drugs 0.000 description 1
- 239000003589 local anesthetic agent Substances 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 241001515942 marmosets Species 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 150000004667 medium chain fatty acids Chemical class 0.000 description 1
- 210000003071 memory t lymphocyte Anatomy 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 108091005601 modified peptides Proteins 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 239000001788 mono and diglycerides of fatty acids Substances 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- MRWXACSTFXYYMV-FDDDBJFASA-N nebularine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC=C2N=C1 MRWXACSTFXYYMV-FDDDBJFASA-N 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000000633 nuclear envelope Anatomy 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 239000007764 o/w emulsion Substances 0.000 description 1
- VQWNELVFHZRFIB-UHFFFAOYSA-N odn 1826 Chemical compound O=C1NC(=O)C(C)=CN1C(O1)CC(O)C1COP(O)(=O)OC1CC(N2C(NC(=O)C(C)=C2)=O)OC1COP(O)(=O)OC1CC(N2C3=C(C(NC(N)=N3)=O)N=C2)OC1COP(O)(=O)OC1CC(N2C(N=C(N)C=C2)=O)OC1COP(O)(=O)OC1CC(N2C3=NC=NC(N)=C3N=C2)OC1COP(O)(=O)OC1CC(N2C3=C(C(NC(N)=N3)=O)N=C2)OC1COP(O)(=O)OC1CC(N2C(NC(=O)C(C)=C2)=O)OC1COP(O)(=O)OC1CC(N2C(N=C(N)C=C2)=O)OC1COP(O)(=O)OC1CC(N2C(N=C(N)C=C2)=O)OC1COP(O)(=O)OC1CC(N2C(NC(=O)C(C)=C2)=O)OC1COP(O)(=O)OC(C(O1)COP(O)(=O)OC2C(OC(C2)N2C3=C(C(NC(N)=N3)=O)N=C2)COP(O)(=O)OC2C(OC(C2)N2C(N=C(N)C=C2)=O)COP(O)(=O)OC2C(OC(C2)N2C3=NC=NC(N)=C3N=C2)COP(O)(=O)OC2C(OC(C2)N2C3=C(C(NC(N)=N3)=O)N=C2)COP(O)(=O)OC2C(OC(C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)OC2C(OC(C2)N2C3=NC=NC(N)=C3N=C2)COP(O)(=O)OC2C(OC(C2)N2C(N=C(N)C=C2)=O)COP(O)(=O)OC2C(OC(C2)N2C(N=C(N)C=C2)=O)COP(O)(=O)OC2C(OC(C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(O)=O)CC1N1C=C(C)C(=O)NC1=O VQWNELVFHZRFIB-UHFFFAOYSA-N 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 239000006174 pH buffer Substances 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 210000005134 plasmacytoid dendritic cell Anatomy 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000002212 purine nucleoside Substances 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 230000031526 regulation of chronic inflammatory response Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- RYYKJJJTJZKILX-UHFFFAOYSA-M sodium octadecanoate Chemical compound [Na+].CCCCCCCCCCCCCCCCCC([O-])=O RYYKJJJTJZKILX-UHFFFAOYSA-M 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 210000000605 viral structure Anatomy 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- JNELGWHKGNBSMD-UHFFFAOYSA-N xanthone Chemical class C1=CC=C2C(=O)C3=CC=CC=C3OC2=C1 JNELGWHKGNBSMD-UHFFFAOYSA-N 0.000 description 1
- 229960001515 yellow fever vaccine Drugs 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
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- 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
- A61P31/14—Antivirals for RNA 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
-
- 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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
- A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
-
- 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
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- the invention is in the field of antiviral treatment, in particular to viral vaccines, and methods for preparing same.
- the present invention provides as an viral vaccine a combination of a protein or peptide antigen and an adjuvant.
- the present invention also provides novel adjuvant combinations.
- the invention also provides methods of treating or preventing viral infectious disease, using the vaccine and adjuvant combinations of the invention.
- BACKGROUND OF THE INVENTION So far, attempts to generate universal vaccines against seasonal viral infections, such as from SARS or influenza virus, have shown limited success due to their failure to stimulate strong T cell responses. This arises from the fact that soluble peptide/protein immunizations are weak inducers of cytotoxic CD8+ T cell (CTL) responses.
- CTL cytotoxic CD8+ T cell
- Most viral vaccines provide protection not by stimulating killer CD8+ T cells against proteins of the virus but by generating neutralizing anti-viral antibodies against surface proteins of the virus (such as the anti-Spike protein in SARS-CoV-2 mRNA vaccines (Pfizer, Moderna) and adenovirus (Astrazeneca)). But RNA immunization also induces only weak T cell responses. Most current influenza virus vaccines also generate neutralizing anti-viral antibodies to target proteins on the surface of the virus that enable entry to host cells (hemagglutinin and neuraminidase).
- Peptide-based vaccines exhibit inefficient co-delivery of antigenic peptides and adjuvants to draining lymph nodes (dLNs), and can have the adverse effect of inducing immunological tolerance and reduced CD8+ T cell immunity.
- typical peptide vaccination protocols include conjugation of the peptides to a carrier protein or TLR agonists or present peptides in a multimeric format for co-delivery with an adjuvant (such multimeric formats include virus-like-particles, nanoparticle or liposomes).
- the antigen would not require special encapsulation such as used in the current SARS-CoV-2 mRNA vaccines (as required in the mRNA vaccines of Pfizer and Moderna) or the need to produce live viruses as the adenovirus vaccines do (Astrazeneca). This would greatly facilitate the production of vaccines, reduce costs and improve safety. It would also be beneficial if the vaccine would not only neutralize the virus and prevent infection as the conventional vaccines above do, but if it could accelerate the clearance of the virus and prevent spreading by killing infected cells before they make more virus particles. Thus there still remains a need to develop strategies that can stimulate potent CTL immunity when using peptides as targets of vaccination. One such strategy may arise from the development of novel adjuvants.
- An adjuvant is an immunopotentiator that is added to enhance the effect of a vaccine.
- the action mechanism of adjuvants has been gradually elucidated.
- various immunoregulatory properties of adjuvants are expected to be applied in the prevention or therapy of not only cancer and infections, but also allergies, cancer, and autoimmune diseases.
- Many vaccine adjuvants have been developed, mainly to induce antibody production (humoral immunity) up to this point.
- Many of the current adjuvants, including alum adjuvants are therefore humoral immunity inducing adjuvants called Th2 adjuvants (type II adjuvants).
- Th2 adjuvants type II adjuvants
- Th1 adjuvants type I adjuvants
- alum aluminium salts
- AS01 contains monophosphoryl lipid A plus saponin fraction QS-21
- AS04 contains alum plus monophosphoryl lipid A
- AS03 contains squalene plus vitamin E
- the present invention now aims to provide a vaccine composition comprising a viral peptide or protein antigen in combination with an adjuvant that allows targeting of proteins of the virus.
- the present invention also aims to provide a vaccine composition comprising a viral peptide or protein antigen in combination with an adjuvant that allows targeting of internal proteins in the virus structure (internal viral proteins), as these proteins are not subject to the same selective pressures during epidemics as viral surface proteins of, for example, SARS-CoV-2 and influenza virus, and thus do not mutate as fast.
- the present invention thus aims to provide a vaccine composition that raises T cell immunity against viral internal proteins so that it can provide a universal vaccine, i.e.
- a vaccine that recognizes and protects against many different strains of the virus, such as against the Delta and Omicron and future variants of SARS- CoV-2 or seasonal variant strains of influenza virus.
- the present inventors have now found a therapeutic and prophylactic antiviral vaccine strategy comprising the use of a viral antigen in a vaccine composition adjuvated with a combination of a CpG oligonucleotide and a STING agonist.
- the inventors herein provide an adjuvant combination of a CpG oligonucleotide and a STING agonist, wherein the CpG oligonucleotide is K3 CpG and wherein the STING agonist is c-di-AMP.
- This adjuvant combination was found to induce a strong CTL immunity against viral protein and peptide antigens.
- the present inventors found that, in mice, a strong CTL immunity was raised against a 20 amino acid immunogenic model peptide antigen (OVA(252-271) (LEQLESIINFEKLTEWTSSN) having the 8 amino acid CTL epitope OVA (257-264) (SIINFEKL)).
- the CTL immunity was induced without the need to employ nanoparticles, which nanoparticles had previously been considered necessary for proper cross-presentation of longer peptides such as the present 20 amino acid antigen when an adjuvant combination of a CpG oligonucleotide and a STING agonist is used.
- an adjuvant combination of a CpG oligonucleotide and a STING agonist unexpectedly circumvents the need to employ nanoparticles to cross-present longer peptides, something that greatly facilitates the production, storage and transport of vaccines for human use.
- mice against a SARS-CoV-2 nucleocapsid (N) protein using a combination of K3 CpG and c- di-AMP as adjuvant were shown, while a strong Th1 and CTL immunity was shown, there was no induction of Th2 immunity (evidenced by absence of IL-5 producing T cells) against the antigens.
- the adjuvant combination of a CpG oligonucleotide and a STING agonist allows the use of viral protein or peptide antigens in a viral vaccine while inducing potent killer CD8+ T cell responses.
- the vaccine compositions of the present generate protection by stimulating killer CD8+ T cells against proteins or peptides of the virus and not by generating neutralizing anti-viral antibodies against surface proteins of the virus.
- the CTL immunity enhancing effect of the combination of K3 CpG and STING agonists is somewhat surprising as STING agonists can inhibit the ability of CpG TLR9 agonists like K3 CpG to induce type I IFN.
- Such type I IFN production is considered a critical signal 3 for the generation of efficient CTL immunity.
- the adjuvant combination offers the advantage that potent anti- viral T cell responses are generated using proteins or peptides. It does not require special encapsulation, conjugation or the need to produce live viruses.
- the present invention provides an antiviral vaccine composition, comprising a viral coat, matrix or core/capsid (glyco)protein as antigen, and an adjuvant combination of a CpG oligonucleotide and a STING agonist, optionally further comprising a pharmaceutically acceptable vehicle, preferably a non-particulate aqueous vehicle.
- the antigen is not a viral surface protein.
- the antigen is an internal viral protein.
- the viral coat, matrix or core/capsid (glyco)protein is from a seasonal virus, including but not limited to coronavirus, influenza virus, respiratory syncytial virus (RSV), norovirus, rhinovirus, parainfluenza, and metapneumovirus, preferably coronavirus, influenza virus, and respiratory syncytial virus (RSV), more preferably Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Influenza A and B virus.
- RSV respiratory syncytial virus
- SARS-CoV Severe Acute Respiratory Syndrome Coronavirus
- Influenza A and B virus may be mammalian or avian variants, preferably human or avian, more preferably human.
- the antigen is a SARS-CoV nucleocapsid (N) protein, preferably SARS-CoV-2 N protein.
- N SARS-CoV nucleocapsid
- CpG oligonucleotide is K3 CpG
- the STING agonist is c-di-AMP.
- the composition is in lyophilized form.
- the present invention provides the antiviral vaccine composition according to the invention as described above, for use in the prevention or treatment of viral infection.
- the present invention provides a method of treating or preventing a viral infection, preferably a SARS infection, more preferably SARS-CoV-2 infection, in a subject in need thereof, comprising administering to said subject a therapeutically or prophylactically effective amount of an antiviral vaccine composition according to the invention as described above.
- the antigen is a SARS nucleocapsid (N) protein, preferably SARS-CoV-2 N protein.
- the CpG oligonucleotide is K3 CpG and the STING agonist is c-di-AMP.
- the present invention provides an antiviral vaccine adjuvant combination comprising K3 CpG and c-di-AMP.
- BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the result of Example 1, wherein mice were vaccinated 3 times with 2-week intervals with SARS-CoV-2 N protein and 10 ⁇ g of K3 CpG and 10 ⁇ g of c-di-AMP or controls, and wherein thirty days after last immunization with, splenocytes were stimulated in vitro with peptide pools (NC pool) that cover the full SARS-CoV-2 N protein.
- NC pool peptide pools
- SARS- CoV-2 N protein plus K3 CpG + c-di-AMP vaccination induces strong memory in both CD4+ and CD8+ T cells as exemplified by the immune response against SARS-CoV-2 N protein of isolated splenocytes stimulated in vitro with peptide pools that cover the full SARS-CoV-2 N protein.
- the immune response was measured as the number of cells producing IFN ⁇ after intracellular cytokine staining and flow cytometry. Each group contained 5 animals.
- Figure 2 shows the amino acid sequence of the nucleocapsid phosphoprotein (N protein), of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Genbank Accession YP_009724397.2.
- FIG. 3 K3 CpG + c-di-AMP adjuvant combination induces strong T cell responses against SARS-CoV-2 nucleocapsid protein (NC) as outlined in Example 3.
- NC SARS-CoV-2 nucleocapsid protein
- FIG. 3 A Time schedule of vaccination. C57BL/6 mice were subcutaneously immunized with 100 ⁇ l of saline, each vaccine containing SARS-CoV-2 nucleocapsid protein (NC) and Addavax, K3 CpG + c-di-AMP, or c-di-AMP alone, or lyophilized vaccine (lyo) containing NC and K3 CpG + c-di-AMP in the flank 3 times every two weeks as indicated the time schedule of vaccination.
- NC SARS-CoV-2 nucleocapsid protein
- mice were immunized subcutaneously with 100 ⁇ l of saline, each vaccine containing SARS-CoV-2 nucleocapsid protein (NC) and Addavax, K3 CpG + c-di-AMP, or c-di-AMP alone, or lyophilized (lyo) vaccine containing NC and K3 CpG + c-di-AMP in the flank 3 times every two weeks as indicated the time schedule of vaccination.
- NC SARS-CoV-2 nucleocapsid protein
- Addavax K3 CpG + c-di-AMP
- c-di-AMP alone
- lyophilized (lyo) vaccine containing NC and K3 CpG + c-di-AMP in the flank 3 times every two weeks as indicated the time schedule of vaccination.
- A, B At 28 days after last vaccine boost (week 8 of experiment), the mice were euthanized and spleens were obtained for analysis.
- CpG oligonucleotide refers to a polynucleotide, preferably an oligonucleotide, comprising at least one non-methylated CG dinucleotide sequence.
- An oligonucleotide comprising at least one CpG motif may comprise multiple CpG motifs.
- CpG motif refers to a non- methylated dinucleotide moiety of an oligonucleotide, comprising a cytosine nucleotide and a subsequent guanosine nucleotide.5-methylcytosine may also be used instead of cytosine.
- a CpG oligonucleotide (CpG ODN) is a short (about 20 base pairs) synthetic single-stranded DNA fragment comprising an immunostimulatory CpG motif.
- a CpG oligonucleotide is a potent agonist of a toll-like receptor 9 (TLR9), which activates dendritic cells (DCs) and B cells to produce type I interferons (IFNs) and inflammatory cytokines (Hemmi, H., et al. Nature 408, 740-745 (2000); Krieg, A. M. Nature reviews. Drug discovery 5, 471-484 (2006).), and acts as an adjuvant of Th1 humoral and cell-mediated immune responses, including cytotoxic T-lymphocyte (CTL) reactions (Brazolot Milian, C. L, Weeratna, R., Krieg, A. M., Siegrist, C. A. & Davis, H. L.
- CTL cytotoxic T-lymphocyte
- CpG ODN has been considered a potential immunotherapeutic agent against inflammation, cancer, asthma, and hay fever (Krieg, A. M. Nature reviews. Drug discovery 5, 471-484 (2006); Klinman, D. M. Nature reviews. Immunology 4, 249-258 (2004)).
- a CpG oligodeoxynucleotide is a synthetic single stranded DNA comprising a non-methylated CpG motif with a immunostimulatory feature due to similarity with a microbial genome, and is recognized by TLR9 in a specific type of natural immune cell [Hartmann et al., J. Immunol. (2000) 164: 944-953; Wagner et al., Trends Immunol. (2004) 25: 1-6].
- TLR9 signals through an adapter molecule myD88 to induce the production of IRF7 dependent type I IFN and NF- ⁇ B dependent cytokines [Krieg et al., Nat. Rev. Drug Discov.
- CpG ODN induces a Th1 response due to the type of cytokine induced by CpG ODN in APC in vivo [Krieg et al., Nat. Rev. Drug Discov. (2006) 5: 471-84].
- type D CpG ODN strongly induces both type I and type II IFN, but cannot induce B cell activation [Krieg et al., Nat. Rev. Drug Discov. (2006) 5: 471-84; Klinman et al., Nat. Rev. Immunol. (2004) 4:1-10].
- Type K CpG ODN (K3 CpG) strongly induces B cell activation to induce IL-6 and antibody production, but they only weakly induce type I and type II IFN.
- type D CpG ODN forms an aggregation, such that only type K CpG can be used for clinical applications [Krieg et al., Nat. Rev. Drug Discov. (2006) 5: 471-84; Klinman et al., Nat. Rev. Immunol. (2004) 4: 1-10].
- Pathogen derived agents such as LPS or non-methylated CpG DNA (CpG) (CpG ODN) stimulate natural immune cells that produce cytokines such as type I or type II IFN and IL-12.
- IL-12 acts on na ⁇ ve CD4 + T cells to derive the generation of Th1 and the production of IFN ⁇ [Seder et al., Proc. Natl. Acad. Sci. U.S.A. (1993) 90: 10188-92; Hsieh et al., Science. (1993) 260: 547-579].
- IFN ⁇ producing Th1 cells are the main actors in the induction of type 1 immunity, which are distinguished by high phagocytic activity [Spellberg et al., Clin. Infect. Dis. (2001) 90509: 76-102; Mantovani et al., Curr. Opin. Immunol. (2010) 22: 231-237]. Furthermore, Th1 cells play an important role in the generation of antitumor immunity and are useful in CTL effector functions and suitable activation including IFN ⁇ production [Hung et al., J. Exp. Med. (1998) 188: 2357-68; Vesely et al., Annu. Rev. Immunol. (2011) 29: 235-271].
- agents, CTLs, and NK cells that can induce a strong Th1 response may play an important role in the development of a vaccine adjuvant or immunotherapeutic agent that is effective against intracellular pathogens or cancer. Therefore, they are in immediate demand. Depending on the difference in backbone modification or surrounding sequences, they are classified into type D/A, type K/B, type C, and type P (Vollmer, J. & Krieg, A. M.
- type D/A induces the production of type I interferon mainly from plasmacytoid dendritic cells (called “plasmacytoid DC” or “pDC”), and type K/B induces B cell growth and the production of IgM, IL-6 or the like.
- Type D/A CpG-DNA strongly induces IFN- ⁇ production, but exhibits low pDC maturation inducing activity and no direct immunostimulatory activity to B cells.
- Type K/B exhibits immunostimulatory activity to B cells, strongly promotes maturation of pDCs, and has high IL-12 inducing capability, but has low IFN- ⁇ inducing capability.
- Type D/A CpG ODN (also called type A, type D or the like and denoted as CpG-A ODN) is an oligonucleotide characterized by a phoshothioate (PS) bond at the 5′ and 3′ terminuses and by a poly G motif with a palindrom (palindromic structure) CpG containing sequence of phosphodiester (PO) in the middle. Cell uptake is facilitated due to the presence of phosphorothioate (PS) at the 5′ and 3′ terminuses.
- PS phoshothioate
- CpG type D/A produces a large quantity of interferon ⁇ (IFN- ⁇ ) in pDCs (different feature from CpG type K/B).
- IFN- ⁇ interferon ⁇
- B cells are not activated and pDCs are not matured (Krug, A., et al. European journal of immunology 31, 2154-2163 (2001).; and Verthelyi, D., et al. Journal of immunology 166, 2372-2377 (2001).)
- Three other types of ODN consist of a PS backbone.
- Type K/B CpG ODN is also called CpG-type B or CpG-type K.
- Type K/B CpG ODN with one or more CpG motifs without a poly G motif have a phosphorothioate (PS) backbone.
- PS phosphorothioate
- type K/B CpG ODN contains multiple CpG motifs with a non-palindromic structure.
- Type K/B CpG has weak IFN- ⁇ inducing activity (produces nearly none), but is a very potent Th1 adjuvant and a potent B cell response stimulating agent which produces IL-6 and activates and matures pDCs (Verthelyi, D., et al. Journal of immunology 166, 2372-2377 (2001); and Hartmann, G. & Krieg, A. M. Journal of immunology 164, 944-953 (2000)).
- Type K/B CpGODN has a function of promoting the survival, activating, and maturing both monocyte derived dendritic cells and pDCs.
- Recently developed type C and type P CpG ODN comprise one and two palindromic structure CpG sequences, respectively. Both can activate B cells, like type K CpG ODN, and activate pDCs, like type D CpG ODN. Meanwhile, type C CpG ODN more weakly induces IFN- ⁇ production relative to type P CpG ODN (Hartmann, G., et al. European journal of immunology 33, 1633-1641 (2003); Marshall, J. D., et al.
- Type D/K and type P CpG ODN are shown to form a higher order structure i.e., Hoogsteen base pair forming a four parallel strand structure called G-tetrads and Watson-Crick base pair between a cis palindromic structure site and a trans palindromic structure site, respectively, which are required for potent IFN- ⁇ production by pDCs (Samulowitz, U., et al. Oligonucleotides 20, 93-101 (2010).; Kerkmann, M., et al.
- type C CpG ODN has a complete phosphorothioate (PS) backbone without a poly G motif, but comprises the type A palindromic sequence of CpG in combination with a stimulatory CpG motif. It is reported from an in vivo study that type C CpG ODN is a very potent Th1 adjuvant.
- PS phosphorothioate
- Type K CpG ODN used in a preferred embodiment in the present invention has a length of 10 nucleotides or longer and comprises the nucleotide sequence set forth in the following formula: 5′-N 1 N 2 N 3 T-CpG-WN 4 N 5 N 6 -3′ [Formula 1] wherein the middle CpG motif (described as CpG) is not methylated, W is A or T, and N1, N2, N3, N4, N5, and N6 may be any nucleotide.
- type K CpG ODN of the invention has a length of 10 nucleotides or longer and comprises the nucleotide sequence of the above-described formula.
- the CpG motif of 4 bases in the middle (TCpGW) only needs to be included in the 10 nucleotides.
- the motif does not necessarily need to be positioned between N3 and N4 in the above-described formula.
- the N1, N2, N3, N4, N5, and N6 may be any nucleotide in the above-described formula.
- Combinations of at least one (preferably one) of N1 and N2, N2 and N3, N3 and N4, N4 and N5, and N5 and N6 may be a two base CpG motif.
- any two contiguous bases in the middle 4 bases (4th to 7th bases) in the above- described formula may be a CpG motif and the other two bases may be any nucleotide.
- a part of or the entire phosphodiester bond of an oligodeoxynucleotide may be substituted with a phosphorothioate bond.
- the entire phosphodiester bond of an oligodeoxynucleotide is substituted with a phosphorothioate bond.
- Type K CpG ODN suitably used in the present invention contains a non-palindromic structure comprising one or more CpG motifs.
- Type K CpG ODN more suitably used in the present invention consists of a non- palindromic structure comprising 1 or more CpG motifs.
- Type K CpG ODN contained in the oligodeoxynucleotide of the invention is preferably humanized. “Humanized” refers to having agonistic activity against human TLR9. Thus, the oligodeoxynucleotide of the invention comprising humanized type K CpG ODN has immunostimulatory activity unique to type K CpG ODN against humans (e.g., activity to activate human B cells to produce IL-6).
- Humanized type K CpG ODN is generally characterized by a four base CpG motif consisting of TCGA or TCGT.
- a single humanized type K CpG ODN comprises 2 or 3 of the four base CpG motifs.
- type K CpG ODN contained in the oligodeoxynucleotide of the invention comprises at least 1, more preferably 2 or more, and still more preferably 2 or 3 four base CpG motifs consisting of TCGA or TCGT.
- these four base CpG motifs may be the same or different. However, this is not particularly limited, as long as there is agonist activity against human TLR9.
- One preferred type K CpG ODN included in the aspects of the invention comprises the nucleotide sequence set forth in the sequence (atcgactctc gagcgttctc (SEQ ID NO: 1)).
- Other suitable CpG ODNs include CpG 1826 (5′- tccatgacgttcctgacgtt-3′ (SEQ ID NO: 2)), D35 CpG (5′-ggtgcatcgatgcagggggg- 3′ (SEQ ID NO: 3)), and the like.
- One particularly preferred type K CpG ODN included in the aspects of the invention comprises the nucleotide sequence set forth in the sequence (atcgactctc gagcgttctc (SEQ ID NO:1)).
- the CpG ODN consists of SEQ ID NO:1 and is herein referred to as K3 CpG.
- the length of type K CpG ODN is not particularly limited, as long as the oligodeoxynucleotide of the invention activates immunostimulatory activity (e.g., activity to activate B cells (preferably human B cells) to produce IL-6) or has anticancer activity, but the length is preferably 100 nucleotides long or less (e.g., 10 to 75 nucleotides long).
- the length of type K CpG ODN is more preferably 50 nucleotides long or less (e.g., 10 to 40 nucleotides long).
- the length of type K CpG ODN is still more preferably 30 nucleotides long or less (e.g., 10 to 25 nucleotides long).
- the length of type K CpG ODN is most preferably 12 to 25 nucleotides long.
- STING ((adapter molecule) stimulator of interferon genes) identified as a membrane protein localized in the endoplasmic reticulum plays an important role in the biological defense mechanism against infections of various RNA viruses and DNA viruses. It is also reported that STING plays an important role in inducing natural immune responses against DNA components derived from microbes and viruses, but the molecular mechanism thereof had not been elucidated.
- STING can form a complex with not only genomic DNA derived from viruses, but also synthetic double stranded DNA of 45 to 90 base pairs called ISD and self-DNA components derived from apoptotic cells. Analysis of DNA interaction region in vitro demonstrated that the C-terminus side region of STING is important. Recognition of various DNA components by STING was demonstrated to induce dynamic local change to regions surrounding the nuclear membrane of STING and to induce interferon production via activation of TBK1. It is also suggested that STING is possibly involved in the regulation of chronic inflammatory responses via recognition of not only allo-DNA component from a microorganism, but also auto-DNA component.
- STING ligand and “STING agonist” are interchangeably used, which is a ligand (agonist) of “STING” ((adapter molecule) stimulator of interferon genes)) inducing type I IFN production and NF- ⁇ B mediated cytokine production.
- STING agonists are considered to be membrane proteins localized in the endoplasmic reticulum.
- cyclic dinucleotides of microbial origin c-di- AMP and c-di-GMP
- STING adapter molecule stimulators of IFN genes
- STING agonist DMXAA
- STING-IRF3 STING-IRF3 mediated type I IFN production
- type 2 immune responses can inhibit a type 1 immune response
- the clinical usefulness of STING agonists including cyclic dinucleotides, was debatable.
- the most common adjuvant, aluminum salt (alum) lacks the ability to induce cell-mediated immunity, which is understood to protect against cancer or diseases from intracellular pathogens [Hogenesch et al., Front. Immunol. (2013) 3: 1-13].
- alums were combined with many different types of adjuvants including monophosphoryl lipid A [Macleod et al., Proc. Natl. Acad. Sci. U.S.A (2011) 108: 7914-7919] and CpG ODN [Weeratna et al., Vaccine. (2000) 18: 1755-1762].
- monophosphoryl lipid A Macleod et al., Proc. Natl. Acad. Sci. U.S.A (2011) 108: 7914-7919
- CpG ODN Weeratna et al., Vaccine. (2000) 18: 1755-1762.
- host DNA may also be a sign of danger as in microorganism DNA, which results in interferon and inflammatory cytokine production [Desmet et al., Nat. Rev. Immunol. (2012) 12: 479-491; Barber et al., Immunol.
- a recently identified cytosol DNA sensor is a cyclic GMP-AMP synthase (cGAS), which catalyzes the production of nonstandard cyclic dinucleotide cGAMP (2′3′-cGAMP) and contains a nonstandard 2′,5′ bond and 3′,5′ bond with the purine nucleoside thereof [Sun et al., Science. (2013) 339: 786-91].
- Standard cGAMP (3′3′) is synthesized in a microbe and has more variety of bonds than mammalian 2′3′-cGAMP.
- STING agonists that can be used in the present invention include cyclic dinucleotides (CDN) such as 2′3′-cGAMP, c-di-AMP, 3′3′-cGAMP, and 3′2′-cGAMP, xanthenone derivatives such as DMXAA, and the like. STING agonists are also explained in WO 2010/017248, whose entire content is incorporated herein by reference.
- the STING agonists is c-di-AMP.
- Cyclic di-adenosine monophosphate c-di-AMP
- an “adjuvant” refers to an immunopotentiator that is added to increase the effect of a vaccine, which is an agent that is not a constituent of a specific antigen but increases immune responses to the administered antigen.
- the term “combination” as used herein is to be understood as referring to simultaneous, separate or sequential administration. In one aspect of the invention “combination” refers to simultaneous administration. In another aspect of the invention “combination” refers to separate administration.
- “combination” refers to sequential administration. Where the administration is sequential or separate, the delay in administering the second component is preferably such that both agents are present in the body so as to produce the effect of the combination.
- a combination of two agents a vaccine and an ICI; a vaccine and an adjuvant; a combination of two agents forming an adjuvant (CpG oligonucleotide and STING agonist) may be administered concomitantly, at different times, as part of the same formulation, as a combination of different formulations, in order, or separately.
- allergy refers to excessive immune responses to a specific antigen.
- allergens from the environment causing allergies are especially called allergens.
- An “allergic disease” refers to a disease induced by an immune response to an exogenous antigen. However, this antigen is often harmless in a quantity that a patient is exposed to in normal life (e.g., pollen during spring time does not have toxicity in and of itself). An immune response resulting in unnecessary discomfort is experienced therewith. This is also called an allergic disease. Examples of typical diseases include atopic dermatitis, allergic rhinitis (hay fever), allergic conjunctivitis, allergic gastroenteritis, bronchial asthma, childhood asthma, food allergy, drug allergy, and hives.
- N protein refers to a viral nucleocapsid protein.
- the nucleic acid and proteins of each class of viruses assemble themselves into a structure called a nucleoprotein, or nucleocapsid.
- the nucleocapsid protein (N protein) is the most abundant protein in coronavirus.
- the N protein is a highly immunogenic phosphoprotein, and it is normally very conserved.
- N protein in aspects of this invention, preferably refers to a coronavirus nucleocapsid protein, still more preferably to a SARS nucleocapsid (N) protein, and most preferably the SARS-CoV-2 N protein as displayed in Figure 2, or sequences having at least 70%, 80%, 90%, 95% or at least 99% sequence similarity with the sequence of Figure 2, determined over the entire length of the protein.
- Cytotoxic T lymphocytes are key players in the immune control of cancer and (viral or bacterial) infection, as they recognize cancer or pathogen derived peptide epitopes presented by HLA class I molecules on the cancer cell or infected cell surface
- the antiviral vaccine strategy provided by the present invention targets the immune system against specific antigens in cancer. It achieves this by combining the viral antigen with a CpG oligonucleotide and a STING agonist as adjuvants.
- the present inventors are the first to show that this adjuvant combination can induce potent CTL responses in vivo.
- the combination of the adjuvants K3 CpG and c-di-AMP is preferred herein.
- the adjuvants and antigen may be used in solution, i.e. they do not need to be presented in the form of nanoparticles.
- Adjuvants and antigen may be used in solution without adjuvants and antigen being conjugated to each other, nor peptides to be conjugated to carrier proteins.
- Adjuvants and antigen in solution do not need to be encapsulated in lipid or viral-like particles.
- this adjuvant combination makes the use of nanoparticles, lipid particles, viral-like particles or conjugations to achieve cross-presentation and immunogenicity redundant.
- the use of the CpG oligonucleotide and a STING agonist combination promotes cross- presentation while stimulating strong CTL immunity.
- cross-presentation refers to the process by which exogenous antigens captured by phagocytic antigen- presenting cells (APCs) are processed and presented onto MHC-I molecules.
- APCs phagocytic antigen- presenting cells
- the vaccine compositions provided herein solves the problem of inducing cross-presentation without the need to generate modified or conjugated peptides or nanoparticles, lipid particles or viral-like particles in order to enhance uptake by APCs. Such modified peptides or nanoparticles would make it much more difficult to produce the vaccine composition under Good Manufacturing Practice (GMP) conditions, due to the chemicals required and complex production process.
- GMP Good Manufacturing Practice
- the presently provided vaccine composition requires only 3 components that can be mixed in aqueous solution, such as saline, and is easy to prepare and administer. These 3 components can be lyophilized together greatly facilitating the storage and transport of the vaccine without the need of a cold chain. Such a lyophilized vaccine can be reconstituted with sterile water just before usage.
- the adjuvant combination described in the present invention can be used with peptides such as synthetic long peptides (e.g., having a length of 10-30 consecutive amino acids) and solves the problem of immunogenicity when using peptides in vaccines.
- Peptide-based vaccines exhibit inefficient co-delivery of antigenic peptides and adjuvants to draining lymph nodes (dLNs), and can have the adverse effect of inducing immunological tolerance and reduced CD8+ T cell immunity.
- conjugate peptides to a carrier protein or TLR agonists or present peptides in a multimeric format for co-delivery with adjuvant include virus-like-particles, nanoparticle or liposomes.
- the adjuvant combination described in the present invention can be used with peptides in solution to induce strong T cell immunity therefore avoiding the need for conjugations and multimeric particles. This is an important advantage as it simplifies manufacturing and safety concerns associated with conjugated molecules and multimeric particles.
- the vaccine compositions of the present invention overcome this problem and allow the use of such relatively long peptides, similarly without exact knowledge of the 8-10 amino acid sequence that is loaded in the MHC- class I complex, whereby efficient cross-presentation of these longer peptides is achieved by using a CpG oligonucleotide and a STING agonist as adjuvants, preferably, the combination of the adjuvants K3 CpG and c-di- AMP.
- K3 CpG may be substituted with humanized K3 CpG.
- Adjuvant combination One highly preferred adjuvant combination of a CpG oligonucleotide and a STING agonist as adjuvants is K3 CpG and c-di-AMP. This adjuvant combination is one aspect of this invention. The adjuvant combination may very suitably be used in combination with cancer neoantigens in (a vaccine for use in) the treatment of cancer. In embodiments, the invention provides the use of a combination of (humanized) K3 CpG oligonucleotide and c-di-AMP as adjuvants together with a short peptide 10-30 amino acid long as antigen.
- the adjuvant combination may very suitably be used in combination with allergen immunotherapy, also known as desensitization or hypo-sensitization.
- allergen immunotherapy also known as desensitization or hypo-sensitization.
- Allergen-specific Immunotherapy (AIT) is the only available treatment aimed to tackle the underlying causes of allergy.
- the active components of subcutaneous vaccines traditionally consist of natural or modified allergen extracts which can be combined with adjuvant platforms.
- the adjuvant platform comprising the combination of K3 CpG and c-di-AMP is provided.
- the adjuvant combination may very suitably be used in combination with antigenic peptides from pathogens in the preparation of a vaccine that can be used to stimulate immunity against viruses, bacteria etc.
- Antiviral vaccine compositions comprising a viral coat, matrix or core/capsid (glyco)protein as antigen, and further comprising as adjuvants a CpG oligonucleotide and a STING agonist.
- a CpG oligonucleotide is K3-CpG and the STING agonist is c-di-AMP.
- the present inventors have found that splenocytes of animals vaccinated with a vaccine comprising a SARS-CoV-2 nucleocapsid (N) protein as antigen and the K3 CpG + c-di-AMP combination as adjuvant, induced very strong IFN ⁇ CD4 and CD8 T cell responses when restimulated with peptides that cover the full length N protein but no Th2 T cell responses. These Th1 and cytotoxic CD8 T responses are considered protective while Th2 responses are considered detrimental (See Examples).
- the antiviral vaccine composition herein provided may also provide protection or treatment of other viral infectious diseases such as influenza (flu) caused by influenza viruses.
- the adjuvant combination of K3 CpG + c-di-AMP may be applied to viral vaccines in general, using any viral coat, matrix or core/capsid (glyco)protein as the antigen.
- the present invention further provides an antiviral vaccine composition comprising the adjuvant combination of K3 CpG + c-di-AMP, wherein the antigen is a SARS nucleocapsid (N) protein, preferably SARS- CoV-2 N protein.
- An antiviral vaccine composition in aspects of the invention comprises a viral coat, matrix or core/capsid (glyco)protein as antigen, and further comprising as adjuvants a CpG oligonucleotide and a STING agonist, preferably the antigen is a viral nucleocapsid protein, more preferably a SARS nucleocapsid (N) protein, even more preferably a SARS- CoV-2 N protein.
- a highly preferred embodiment of the CpG oligonucleotide and a STING agonist combination is in aspects of this invention is the combination K3 CpG + c-di-AMP.
- the present invention also provides a method of treating or preventing a viral infection, preferably a SARS infection, more preferably SARS-CoV-2 infection, comprising administrating to a subject in need thereof a therapeutically or prophylactically effective amount of a vaccine composition as described above comprising a viral coat, matrix or core/capsid (glyco)protein as the antigen.
- Medicaments and Dosage Forms The present invention is provided as medicaments (therapeutic agent or prophylactic agent) in various forms described above.
- the route of administration of a therapeutic agent, prophylactic agent, or the like that is effective upon therapy is preferably used, such as intravenous, subcutaneous, intramuscular, intraperitoneal, oral administration, or the like.
- dosage form examples include injection, capsules, tablets, granules, and the like.
- the components of the present invention are effectively used upon administration as an injection.
- Aqueous solutions for injection may be stored, for example, in a vial or a stainless steel container.
- Aqueous solutions for injections may also be blended with, for example, saline, sugar (e.g., trehalose), NaCl, NaOH, or the like.
- Therapeutic agents may also be blended, for example, with a buffer (e.g., phosphate buffer), stabilizer, or the like.
- the composition, medicament, therapeutic agent, prophylactic agent, or the like of the present invention comprises a therapeutically effective amount of a therapeutic agent or effective ingredient, and a pharmaceutically acceptable carrier or excipient.
- carrier refers to a diluent, adjuvant, excipient or vehicle administered with a therapeutic agent.
- a carrier can be an aseptic liquid such as water or oil, including, but not limited to, those derived from petroleum, animal, plant or synthesis, as well as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
- water is a preferred carrier.
- saline and aqueous dextrose are preferred carriers.
- an aqueous saline solution and aqueous dextrose and glycerol solution are used as a liquid carrier of an injectable solution.
- Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, powdered skim milk, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylamino acetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, saccharose, carboxymethylcellulose, corn starch, inorganic salt, and the like.
- the composition can also contain a small amount of wetting agent, emulsifier, or pH buffer.
- these compositions can be in a form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release preparation, or the like.
- traditional binding agents and carriers such as triglyceride
- Oral preparation can also comprise a standard carrier such as medicine grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, or magnesium carbonate. Examples of a suitable carrier are described in E. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A.).
- Such a composition contains a therapeutically effective amount of therapy agent, preferably in a purified form, together with a suitable amount of carrier, such that the composition is provided in a form suitable for administration to a patient.
- a preparation must be suitable for the administration format.
- the composition may comprise, for example, a surfactant, excipient, coloring agent, flavoring agent, preservative, stabilizer, buffer, suspension, isotonizing agent, binding agent, disintegrant, lubricant, fluidity improving agent, corrigent, or the like.
- salt in one embodiment of the present invention include anionic salts formed with any acidic (e.g., carboxyl) group and cationic salts formed with any basic (e.g., amino) group.
- Salts include inorganic salts and organic salts, as well as salts described in, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19. Examples thereof further include metal salts, ammonium salts, salts with organic base, salts with inorganic acid, salts with organic acid, and the like.
- “Solvate” in one embodiment of the present invention is a compound formed with a solute or solvent.
- solvates When a solvent is water, a solvate formed thereof is a hydrate. It is preferable that the solvent does not obstruct the biological activity of the solute.
- Examples of such a preferred solvent include, but not particularly limited to, water and various buffers.
- Examples of “chemical modification” in one embodiment of the present invention include modifications with PEG or a derivative thereof, fluorescein modification, biotin modification, and the like.
- Examples of such a system include use of a recombinant cell that can express encapsulated therapeutic agent (e.g., polypeptide) in liposomes, microparticles, and microcapsules; use of endocytosis mediated by a receptor; construction of a therapy nucleic acid as a part of a retrovirus vector or another vector; and the like.
- encapsulated therapeutic agent e.g., polypeptide
- a medicament can be administered by any suitable route, such as by injection, bolus injection, or by absorption through epithelial or mucocutaneous lining (e.g., oral cavity, rectum, intestinal mucosa, or the like).
- an inhaler or mistifier using an aerosolizing agent can be used as needed.
- other biological activating agents can also be administered concomitantly. Administration can be systemic or local.
- the present invention can be administered by any suitable route such as direct injection into cancer (lesion).
- a composition can be prepared as a pharmaceutical composition adapted to administration to humans in accordance with a known method. Such a composition can be administered by an injection.
- a composition for injection is typically a solution in an aseptic isotonic aqueous buffer.
- a composition can also comprise a local anesthetic such as lidocaine, which alleviates the pain at the site of injection, and a solubilizing agent as needed.
- ingredients can be supplied individually or by mixing the ingredients together in a unit dosage form; and supplied, for example, in a sealed container such as an ampoule or sachet showing the amount of active agent or as a lyophilized powder or water-free concentrate.
- the composition can be distributed using an injection bottle containing aseptic agent-grade water or saline.
- composition When composition is to be administered by injection, an aseptic water or saline ampoule for injection can also be provided such that the ingredients can be mixed prior to administration.
- the composition, medicament, therapeutic agent, and prophylactic agent of the invention can be prepared as a neutral or base form or other prodrugs (e.g., ester or the like).
- Pharmaceutically acceptable salts include salts formed with a free carboxyl group, derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, or the like, salts formed with a free amine group, derived from isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, or the like; and salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide or the like.
- the amount of therapeutic agent of the invention that is effective in therapy of a specific disorder or condition may vary depending on the properties of the disorder or condition. However, such an amount can be determined by those skilled in the art with a standard clinical technique based on the descriptions herein.
- an in vitro assay can be used in some cases to assist the identification of the optimal dosing range.
- the precise dose to be used for a preparation may also vary depending on the route of administration or the severity of the disease or disorder. Thus, the dose should be determined in accordance with the judgment of the attending physician or the condition of each patient.
- the dosage is not particularly limited, but may be 0.001, 1, 5, 10, 15, 100, or 1000 mg/kg body weight per dosage or within a range between any two values described above.
- the dosing interval is not particularly limited, but may be, for example, 1 or 2 doses every 1, 7, 14, 21, or 28 days or 1 or 2 doses in a range of period between any two values described above.
- the dosage, dosing interval, and dosing method may be appropriately selected depending on the age, weight, symptom, target organ, or the like of the patient. Further, it is preferable that a therapeutic agent contains a therapeutically effective amount of effective ingredients, or an amount of effective ingredients effective for exerting a desired effect. When a malignant tumor marker significantly decreases after administration, the presence of a therapeutic effect may be acknowledged.
- the effective dose can be estimated from a dose-response curves obtained from in vitro or animal model testing systems.
- the amount of N protein per vaccine injection may be in the range of about 100 ⁇ g to 10mg, preferably about 500 ⁇ g to 5mg, or more preferably about 1mg to 3mg, or about 2mg.
- the amount of each adjuvant in the adjuvant combination comprised in a single dose vaccine injection is preferably about or equal in weight.
- the ratio between the ODN and STING agonist in an adjuvant combination of the invention may be 1:5 or 5:1, preferably the weight ratio is 1:2 to 2:1. Most preferably, the weight ratio is about 1:1.
- the amount of each adjuvant may be in the range of 100 ⁇ g to 3mg.
- an amount of K3 CpG of 100 ⁇ g, 500 ⁇ g, 1mg, 2mg or 3mg may suitably be combined with an amount of c-di-AMP of 100 ⁇ g, 500 ⁇ g, 1mg, 2mg or 3mg per vaccine injection.
- “Patient” or “subject” in one embodiment of the present invention includes humans and mammals excluding humans (e.g., one or more species of mice, guinea pigs, hamsters, rats, rabbits, pigs, sheep, goats, cows, horses, cats, dogs, marmosets, monkeys, and the like).
- the pharmaceutical composition, therapeutic agent, or prophylactic agent of the invention can be provided as a kit.
- the present invention provides an agent pack or kit comprising one or more containers filled with one or more ingredients of the composition or medicament of the invention.
- the pharmaceutical composition comprising an ingredient of the present invention can be administered via liposomes, microparticles, or microcapsules.
- the formulation procedure for the therapeutic agent, prophylactic agent, or the like of the invention as a medicament or the like is known in the art.
- SARS-CoV-2 N protein plus K3 CpG + c-di-AMP vaccination induces strong memory in both CD4+ and CD8+ T cells
- Vaccination with SARS-CoV-2 nucleocapsid (N) protein as antigen and the K3 CpG + c-di-AMP combination as adjuvant induces potent CD4 and CD8 T cell memory responses.
- Mice were vaccinated 3 times with 2- week intervals and 30 days after last immunization they were euthanized and the immune response against SARS-CoV-2 N protein was tested. Animals were vaccinated with 3 ⁇ g of SARS-CoV-2 N protein and 10 ⁇ g of K3 CpG and 10 ⁇ g of c-di-AMP.
- SARS CoV-2 N protein plus K3 CpG + c-di-AMP vaccination induces strong Th1 and CD8 T cell memory but no Th2 memory Vaccination with SARS-CoV-2 nucleocapsid (N) protein as antigen and the K3 CpG + c-di-AMP combination as adjuvant, induces potent IFN ⁇ - producing Th1 CD4 and CD8 T cell memory responses with no Th2 (IL-5 producing) responses.
- Mice were vaccinated 3 times with 2-week intervals and 30 days after last immunization they were euthanized and the immune response against SARS-CoV-2 N protein was tested.
- mice were vaccinated with 3 ⁇ g of SARS-CoV-2 N protein and 10 ⁇ g of K3 CpG and 10 ⁇ g of c-di-AMP. Thirty days after last immunization, the time at which memory immunity is established, splenocytes were stimulated in vitro in an ELISpot assay with peptide pools that cover the full SARS-CoV-2 N protein. IFN ⁇ - or IL-5 producing T cells were measured by ELISpot assay. Each group 7-8 animals. Results are displayed in Table 1. Table 1. IFN ⁇ - or IL-5 producing T cells as measured by ELISpot assay.
- SARS-CoV-2 nucleocapsid protein (N protein; C- terminal His tagged full length protein of YP_009724397.2) was purchased from GeneTex, Inc. Irvine, CA, USA.
- SARS-CoV-2 nucleoprotein peptide pool JPT PepMix SARS-CoV-2 (NCAP; 10215-mer peptides with 11 amino acid overlap) was purchased from JPT (JPT Peptide Technologies GmbH, Berlin, Germany).
- Adjuvants used were K3 CpG ((ATCG ACTC TCGA GCGT TCTC); GeneDesign Inc., Osaka, Japan) and c-di-AMP (CAS: 54447-84-6; Yamasa Corporation, Chiba, Japan).
- AddaVax TM (InVivoGen, San Diego, USA) squalene-based commercial adjuvant was included as a control in some immunizations.
- SARS-CoV-2 nucleoprotein vaccinations In order to test the SARS-CoV-2 nucleoprotein vaccine, mice were vaccinated subcutaneously in the left flank 3 times, once every two weeks.
- mice Thirty days after the last immunization mice were euthanized, and blood, spleen and left axillary and inguinal lymph nodes were collected for in vitro measurement of the memory T cell immune response.
- Mice were vaccinated with 100 ⁇ l volume that contained 3 ⁇ g of SARS-CoV-2 nucleoprotein, 10 ⁇ g of K3 CpG and 10 ⁇ g c-di-AMP adjuvants in sterile saline.
- the same amount of protein was injected in 100 ⁇ l sterile saline or 100 ⁇ l of a mix of equal volumes of sterile saline and AddaVax TM . Flow cytometry.
- Single-cell suspensions of splenocytes and lymph nodes were generated by mechanical disruption of spleens and lymph nodes and then filtering through a 40 ⁇ m cell strainer (Falcon, San Jose, CA, USA). Cells were washed and counted, 2x10 6 cells were used for staining. For blood staining 60 ⁇ l of blood were used. After lysing erythrocytes, cells were washed and stained. In all stains cells were first pre-treated with Fc block for 10 min. For surface stains, cells were stained for 20 min on ice with different panels of antibodies.
- HBSS Hank's Balanced Salt Solution
- FBS fetal bovine serum
- PFA paraformaldehyde
- cytokine staining cells were stimulated with 10 ⁇ g/ml SARS-CoV-2 nucleoprotein peptide pool for 6 hrs at 37°C in 5% CO 2 in the presence of GolgiPlug TM (BD Biosciences) and CD107a-APC-Cy7 antibody.
- Cells were fixed overnight with IC Fixation Buffer at 4°C, washed with a Perm/Wash buffer (both from eBiosciences, Thermo Fisher Scientific, Waltham, MA, USA) and stained for intracellular cytokines for 45 min at 4°C. Fluorochrome conjugated antibodies for CD4 and CD8 were used. Finally, cells were washed twice with Perm/Wash buffer and fixed with 1% PFA.
- PMA and Ionomycin were used as a positive control.
- Anti-murine detection antibody was added and incubated at room temperature for 2 hours. After washing 3 times the plates with PBS Tween 0.05%, Streptavidin solution was added and incubated at room temperature for 30min.
- Vaccination with SARS-CoV-2 NC together K3 CpG plus c-di-AMP adjuvant combination elicits strong memory Th1 immunity without memory Th2 immunity against NC protein which is elicited when c-di-AMP is used alone as an adjuvant Figure 4A.
- Lyophilized vaccine containing SARS-CoV-2 NC together with K3 CpG plus c-di-AMP adjuvant combination retains its activity ( Figures 3 and 4). This is important as the vaccine does not require a cold chain for transport.
- SARS-CoV-2 nucleocapsid protein NC (GeneTex) was used for immunization.
- TLR9 agonist K type of CpG ODN (K3 CpG) and STING agonist c-di-AMP adjuvants were used in vivo at 10 ⁇ g per injection in sterile saline.
- K3 CpG was synthesized by GeneDesign (Japan) and c-di- AMP was kindly provided by Yamasa (Japan).
- AddavaxTM (InVivoGen, USA) was used by mixing it with an equal volume of sterile saline. In some experiments lyophilization of vaccines was tested. Vaccines containing SARS-CoV-2 nucleocapsid protein (NC) and K3 CpG + c-di-AMP were frozen in a -80°C freezer overnight and next day they were lyophilized by a Freeze- dryer Alpha 1-2 LDplus (CHRIST) overnight.
- NC SARS-CoV-2 nucleocapsid protein
- K3 CpG + c-di-AMP were frozen in a -80°C freezer overnight and next day they were lyophilized by a Freeze- dryer Alpha 1-2 LDplus (CHRIST) overnight.
- C57BL/6 mice were immunized with 100 ⁇ l of saline, each vaccine containing (3 ⁇ g of) SARS- CoV-2 nucleocapsid protein (NC) and one of Addavax (3 ⁇ g of NC in Addavax), K3 CpG + c-di-AMP, or c-di-AMP, or lyophilized and water- reconstituted vaccine, containing 3 ⁇ g of NC and 10 ⁇ g K3 CpG + 10 ⁇ g c-di- AMP. Immunization occurred in the flank on days 0, 14, and 21. On days 21 and 35, blood were obtained for ELISpot analysis.
- NC SARS- CoV-2 nucleocapsid protein
- Addavax 3 ⁇ g of NC in Addavax
- K3 CpG + c-di-AMP c-di-AMP
- lyophilized and water- reconstituted vaccine containing 3 ⁇ g of NC and 10 ⁇ g K3 CpG + 10 ⁇ g c-
- mice were euthanized and blood and spleens were obtained for analysis.
- Flow cytometry For flow cytometry measurement of NC-specific CD4+ and CD8+ T cells, single-cell suspensions of splenocytes, tissue were mechanically disrupted and filtered through a 40 ⁇ m cell strainer (Falcon, San Jose, CA, USA). Cells were washed and counted, and 2x10 6 cells were used for staining. For staining, cells were first pre-treated with anti-CD16/CD32 blocking antibodies for 10 min. For surface stains, cells were stained for 20 min on ice with surface staining antibodies.
- cells were stained with PerCP-Cy 5.5-labeled Annexin V (BD Biosciences, San Jose, CA, USA) After staining, cells were washed with HBSS containing 3% FBS and 0.02% sodium azide and fixed with 1% PFA. When Annexin V was used, all buffers contained 2.5 mM of CaCl2. For intracellular cytokine staining, cells were stimulated with 2 ⁇ g/ml PepMix SARS-CoV-2 (NCAP) (JPT, see above) for 18 hrs at 37°C in 5% CO2 in the presence of GolgiPlug (BD Bioscience) and anti-CD107a-APC-Cy7 antibody.
- NCAP PepMix SARS-CoV-2
- NC-specific T cells For measuring NC-specific T cells by ELISPOT assay, plates pre- coated with anti-murine IFN- ⁇ antibody or anti-murine IL-5 antibody were used (ImmunoSpot – CTL, USA). The protocol followed was as the manufacturer recommended. Briefly, 1x10 5 cells for IFN- ⁇ plates and 4x10 5 cells for IL-5 plates were seeded in 100 ⁇ l per well, and 100 ⁇ l of media, PepMix SARS-CoV-2 (NCAP) (JPT) at 2 ⁇ g/ml were used to stimulate cells. Cells were incubated overnight at 37°C in 7% CO2. The next day the wells were washed twice with PBS and twice with PBS Tween 0.05%.
- NCAP PepMix SARS-CoV-2
- JPT PepMix SARS-CoV-2
- Anti-murine detection antibody was added and incubated at RT for 2 hrs. After washing three times the plates with PBS Tween 0.05%, Streptavidin-AP solution was added and incubated at RT for 30 min. The plates were washed two more times with PBS Tween 0.05% and two times with deionized H2O and the developer solution was added and incubated at RT for 15 min. The reaction was stopped by washing the plates with water, afterwards they were allowed to air-dry for at least 24 hours before reading. The plates were read and analyzed in a CTL counter with ImmunoSpot Software (USA).
- a negative control to test specificity of vaccine protection included lyophilized 3 ⁇ g full length ovalbumin protein (OVA) plus 10 ⁇ g of K3 CpG and 10 ⁇ g of c-di-AMP in 100 ⁇ l of sterile saline. Mice were followed up to day 14 post viral challenge for weight loss and survival.
- OVA ovalbumin protein
Abstract
The present invention provides an antiviral vaccine composition, comprising a viral coat, matrix or core/capsid (glyco)protein as antigen, and an adjuvant combination of a CpG oligonucleotide and a STING agonist.
Description
Title: Antiviral vaccine composition FIELD OF THE INVENTION The invention is in the field of antiviral treatment, in particular to viral vaccines, and methods for preparing same. The present invention provides as an viral vaccine a combination of a protein or peptide antigen and an adjuvant. The present invention also provides novel adjuvant combinations. The invention also provides methods of treating or preventing viral infectious disease, using the vaccine and adjuvant combinations of the invention. BACKGROUND OF THE INVENTION So far, attempts to generate universal vaccines against seasonal viral infections, such as from SARS or influenza virus, have shown limited success due to their failure to stimulate strong T cell responses. This arises from the fact that soluble peptide/protein immunizations are weak inducers of cytotoxic CD8+ T cell (CTL) responses. Most viral vaccines provide protection not by stimulating killer CD8+ T cells against proteins of the virus but by generating neutralizing anti-viral antibodies against surface proteins of the virus (such as the anti-Spike protein in SARS-CoV-2 mRNA vaccines (Pfizer, Moderna) and adenovirus (Astrazeneca)). But RNA immunization also induces only weak T cell responses. Most current influenza virus vaccines also generate neutralizing anti-viral antibodies to target proteins on the surface of the virus that enable entry to host cells (hemagglutinin and neuraminidase). One example of a successful viral vaccine is the Yellow Fever vaccine that elicits CTL responses of ~2-10% and provides lifelong protection (Akondy RS, et al.2017 Nature 52(7685):362-367). Thus if clinically relevant T cell responses to viral
antigens are to be achieve, there is a need for vaccine strategies that stimulate more T cell immunity. Proteins and peptides on their own are poor inducers of T cell immunity. This is a general problem relating to the immunogenicity of peptides or proteins when they are used in vaccines. Peptide-based vaccines exhibit inefficient co-delivery of antigenic peptides and adjuvants to draining lymph nodes (dLNs), and can have the adverse effect of inducing immunological tolerance and reduced CD8+ T cell immunity. To solve this problem, typical peptide vaccination protocols include conjugation of the peptides to a carrier protein or TLR agonists or present peptides in a multimeric format for co-delivery with an adjuvant (such multimeric formats include virus-like-particles, nanoparticle or liposomes). It would be beneficial if the antigen would not require special encapsulation such as used in the current SARS-CoV-2 mRNA vaccines (as required in the mRNA vaccines of Pfizer and Moderna) or the need to produce live viruses as the adenovirus vaccines do (Astrazeneca). This would greatly facilitate the production of vaccines, reduce costs and improve safety. It would also be beneficial if the vaccine would not only neutralize the virus and prevent infection as the conventional vaccines above do, but if it could accelerate the clearance of the virus and prevent spreading by killing infected cells before they make more virus particles. Thus there still remains a need to develop strategies that can stimulate potent CTL immunity when using peptides as targets of vaccination. One such strategy may arise from the development of novel adjuvants. An adjuvant is an immunopotentiator that is added to enhance the effect of a vaccine. With the recent development in immunology, the action mechanism of adjuvants has been gradually elucidated. Recently, various immunoregulatory properties of adjuvants are expected to be applied in the prevention or therapy of not only cancer and infections, but also allergies, cancer, and autoimmune diseases.
Many vaccine adjuvants have been developed, mainly to induce antibody production (humoral immunity) up to this point. Many of the current adjuvants, including alum adjuvants are therefore humoral immunity inducing adjuvants called Th2 adjuvants (type II adjuvants). However, induction of cell-mediated immunity is more important than humoral immunity in the prevention or therapy of cancer or allergies. Such adjuvants are called Th1 adjuvants (type I adjuvants). Currently only 6 adjuvants have been approved for human use. These adjuvants are used with proteins or heat inactivated viruses (basically the proteins of the virus). These include aluminium salts (alum), oil-in-water emulsion squalene adjuvant MF59, AS01 (contains monophosphoryl lipid A plus saponin fraction QS-21), AS04 (contains alum plus monophosphoryl lipid A), AS03 (contains squalene plus vitamin E) and oligodeoxynucleotides CpG 1018. These adjuvants have been designed primarily to induce antibody-mediated protection and vary in their ability to induce CD4+ T cell immunity (Del Giudice, G., et al.2018. Semin Immunol 39, 14-21; Pulendran, B., et al.2021. Nat Rev Drug Discov 20, 454-475). Critically, the ability of these adjuvants to induce killer CD8+ T cell responses is much lower compared to viral vaccines (Akondy, R.S., et al. 2017. Nature 552, 362-367; Miller, J.D., et al.2008 Immunity 28, 710-722; Akondy, R.S., et al.2015. Proc Natl Acad Sci USA 112, 3050-3055). The present invention now aims to provide a vaccine composition comprising a viral peptide or protein antigen in combination with an adjuvant that allows targeting of proteins of the virus. The present invention also aims to provide a vaccine composition comprising a viral peptide or protein antigen in combination with an adjuvant that allows targeting of internal proteins in the virus structure (internal viral proteins), as these proteins are not subject to the same selective pressures during epidemics as viral surface proteins of, for example, SARS-CoV-2 and influenza virus, and thus do not mutate as fast. The present invention thus
aims to provide a vaccine composition that raises T cell immunity against viral internal proteins so that it can provide a universal vaccine, i.e. a vaccine that recognizes and protects against many different strains of the virus, such as against the Delta and Omicron and future variants of SARS- CoV-2 or seasonal variant strains of influenza virus. SUMMARY OF THE INVENTION The present inventors have now found a therapeutic and prophylactic antiviral vaccine strategy comprising the use of a viral antigen in a vaccine composition adjuvated with a combination of a CpG oligonucleotide and a STING agonist. In particular, the inventors herein provide an adjuvant combination of a CpG oligonucleotide and a STING agonist, wherein the CpG oligonucleotide is K3 CpG and wherein the STING agonist is c-di-AMP. This adjuvant combination was found to induce a strong CTL immunity against viral protein and peptide antigens. The present inventors found that, in mice, a strong CTL immunity was raised against a 20 amino acid immunogenic model peptide antigen (OVA(252-271) (LEQLESIINFEKLTEWTSSN) having the 8 amino acid CTL epitope OVA(257-264) (SIINFEKL)). Unexpectedly, the CTL immunity was induced without the need to employ nanoparticles, which nanoparticles had previously been considered necessary for proper cross-presentation of longer peptides such as the present 20 amino acid antigen when an adjuvant combination of a CpG oligonucleotide and a STING agonist is used. Thus the use of the adjuvant combination of a CpG oligonucleotide and a STING agonist unexpectedly circumvents the need to employ nanoparticles to cross-present longer peptides, something that greatly facilitates the production, storage and transport of vaccines for human use. Also, as outlined in the experimental part herein below, the same potent in vivo T cell immune response was observed in mice against a
SARS-CoV-2 nucleocapsid (N) protein using a combination of K3 CpG and c- di-AMP as adjuvant. Importantly, while a strong Th1 and CTL immunity was shown, there was no induction of Th2 immunity (evidenced by absence of IL-5 producing T cells) against the antigens. The adjuvant combination of a CpG oligonucleotide and a STING agonist allows the use of viral protein or peptide antigens in a viral vaccine while inducing potent killer CD8+ T cell responses. The vaccine compositions of the present generate protection by stimulating killer CD8+ T cells against proteins or peptides of the virus and not by generating neutralizing anti-viral antibodies against surface proteins of the virus. The CTL immunity enhancing effect of the combination of K3 CpG and STING agonists is somewhat surprising as STING agonists can inhibit the ability of CpG TLR9 agonists like K3 CpG to induce type I IFN. Such type I IFN production is considered a critical signal 3 for the generation of efficient CTL immunity. The adjuvant combination offers the advantage that potent anti- viral T cell responses are generated using proteins or peptides. It does not require special encapsulation, conjugation or the need to produce live viruses. This greatly facilitates the production of vaccines, and further provides the ability to freeze dry the vaccine, meaning that cold-chain from the vaccine manufacturer to the vaccination site is no longer required. A freeze dried vaccine can be shipped and stored at room temperature, reducing transport and storage costs and increasing availability of the vaccine. In a first aspect, the present invention provides an antiviral vaccine composition, comprising a viral coat, matrix or core/capsid (glyco)protein as antigen, and an adjuvant combination of a CpG oligonucleotide and a STING agonist, optionally further comprising a
pharmaceutically acceptable vehicle, preferably a non-particulate aqueous vehicle. In a preferred embodiment of an antiviral vaccine composition according to the present invention the antigen is not a viral surface protein. In another preferred embodiment of an antiviral vaccine composition according to the present invention the antigen is an internal viral protein. In another preferred embodiment of an antiviral vaccine composition according to the present invention the viral coat, matrix or core/capsid (glyco)protein is from a seasonal virus, including but not limited to coronavirus, influenza virus, respiratory syncytial virus (RSV), norovirus, rhinovirus, parainfluenza, and metapneumovirus, preferably coronavirus, influenza virus, and respiratory syncytial virus (RSV), more preferably Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Influenza A and B virus. These viruses may be mammalian or avian variants, preferably human or avian, more preferably human. In another preferred embodiment of an antiviral vaccine composition according to the present invention the antigen is a SARS-CoV nucleocapsid (N) protein, preferably SARS-CoV-2 N protein. In another preferred embodiment of an antiviral vaccine composition according to the present invention CpG oligonucleotide is K3 CpG, and the STING agonist is c-di-AMP. In another preferred embodiment of an antiviral vaccine composition according to the present invention the composition is in lyophilized form. In another aspect, the present invention provides the antiviral vaccine composition according to the invention as described above, for use in the prevention or treatment of viral infection. In another aspect, the present invention provides a method of treating or preventing a viral infection, preferably a SARS infection, more
preferably SARS-CoV-2 infection, in a subject in need thereof, comprising administering to said subject a therapeutically or prophylactically effective amount of an antiviral vaccine composition according to the invention as described above. In a preferred embodiment of a method of treating or preventing a viral infection according to the present invention, the antigen is a SARS nucleocapsid (N) protein, preferably SARS-CoV-2 N protein. In another preferred embodiment of a method of treating or preventing a viral infection according to the present invention, the CpG oligonucleotide is K3 CpG and the STING agonist is c-di-AMP. In another aspect, the present invention provides an antiviral vaccine adjuvant combination comprising K3 CpG and c-di-AMP. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the result of Example 1, wherein mice were vaccinated 3 times with 2-week intervals with SARS-CoV-2 N protein and 10 µg of K3 CpG and 10 µg of c-di-AMP or controls, and wherein thirty days after last immunization with, splenocytes were stimulated in vitro with peptide pools (NC pool) that cover the full SARS-CoV-2 N protein. SARS- CoV-2 N protein plus K3 CpG + c-di-AMP vaccination induces strong memory in both CD4+ and CD8+ T cells as exemplified by the immune response against SARS-CoV-2 N protein of isolated splenocytes stimulated in vitro with peptide pools that cover the full SARS-CoV-2 N protein. The immune response was measured as the number of cells producing IFNγ after intracellular cytokine staining and flow cytometry. Each group contained 5 animals. Figure 2 shows the amino acid sequence of the nucleocapsid phosphoprotein (N protein), of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Genbank Accession YP_009724397.2.
Figure 3: K3 CpG + c-di-AMP adjuvant combination induces strong T cell responses against SARS-CoV-2 nucleocapsid protein (NC) as outlined in Example 3. (A) Time schedule of vaccination. C57BL/6 mice were subcutaneously immunized with 100 µl of saline, each vaccine containing SARS-CoV-2 nucleocapsid protein (NC) and Addavax, K3 CpG + c-di-AMP, or c-di-AMP alone, or lyophilized vaccine (lyo) containing NC and K3 CpG + c-di-AMP in the flank 3 times every two weeks as indicated the time schedule of vaccination. (B and C) IFN-γ ELISpot using PBMCs. On day 21 (week 3), day 35 (week 5), and day 56 (week 8), blood was obtained for ELISpot analysis. The number of NC-specific T cells producing IFN-γ were measured by ELISpot after 24 hours of restimulation of PBMCs with NC protein peptide mix. Each dot represents an individual mouse. SPFC/SFC: spot forming cells. Figure 4: K3 CpG + c-di-AMP adjuvant combination induces potent memory CD4+ and CD8+ T cell immunity against SARS-CoV-2 nucleocapsid protein (NC) as outlined in Example 3. C57BL/6 mice were immunized subcutaneously with 100 µl of saline, each vaccine containing SARS-CoV-2 nucleocapsid protein (NC) and Addavax, K3 CpG + c-di-AMP, or c-di-AMP alone, or lyophilized (lyo) vaccine containing NC and K3 CpG + c-di-AMP in the flank 3 times every two weeks as indicated the time schedule of vaccination. (A, B) At 28 days after last vaccine boost (week 8 of experiment), the mice were euthanized and spleens were obtained for analysis. To assess Th1 and Th2 immunity against SARS-CoV-2 nucleocapsid protein, the number of NC-specific T cells producing IFN-γ and IL-5 were measured by ELISpot after 24 hrs of restimulation of splenocytes with NC protein peptide mix (A). The frequencies of IFN-γ producing NC- specific CD8+ and CD4+ T cells were analyzed by intracellular staining after restimulation with NC protein (B). Each dot represents an individual mouse. SFC: spot forming cells.
Figure 5: A vaccine including SARS-CoV-2 nucleocapsid protein (NC) and K3 CpG + c-di-AMP adjuvant combination protects mice against SARS-CoV-2 viral challenge as outlined in Example 3. A) Vaccination and viral challenge schedule in K18-hACE2 transgenic mice. B) Survival curves for mice vaccinated and then intranasally challenged with 104 PFU SARS- CoV2 Wuhan strain (Wu-Hu-1) 50µl in DMEM. Only mice vaccinated with SARS-CoV-2 NC protein plus K3 CpG and c-di-AMP (NC/K3/c-di-AMP) in 100 µl of sterile saline are protected. Unvaccinated (saline vaccination) controls animals or animals vaccinated with SARS-CoV-2 NC protein in AddaVax (NC/Addavax) were not protected. Animals vaccinated with ovalbumin protein (OVA) plus K3 CpG and c-di-AMP (OVA/K3/c-di-AMP) were also not protected. DETAILED DESCRIPTION OF THE INVENTION The definition of the terms and/or general techniques particularly used herein is explained hereinafter, as appropriate. As used herein, “CpG oligonucleotide”, “CpG oligodeoxynucleotide”, “CpG ODN”, or “simply “CpG” are interchangeably used, and refer to a polynucleotide, preferably an oligonucleotide, comprising at least one non-methylated CG dinucleotide sequence. An oligonucleotide comprising at least one CpG motif may comprise multiple CpG motifs. As used herein, the phrase “CpG motif” refers to a non- methylated dinucleotide moiety of an oligonucleotide, comprising a cytosine nucleotide and a subsequent guanosine nucleotide.5-methylcytosine may also be used instead of cytosine. A CpG oligonucleotide (CpG ODN) is a short (about 20 base pairs) synthetic single-stranded DNA fragment comprising an immunostimulatory CpG motif. A CpG oligonucleotide is a potent agonist of a toll-like receptor 9 (TLR9), which activates dendritic cells (DCs) and B cells to produce type I interferons (IFNs) and inflammatory cytokines (Hemmi, H., et al. Nature
408, 740-745 (2000); Krieg, A. M. Nature reviews. Drug discovery 5, 471-484 (2006).), and acts as an adjuvant of Th1 humoral and cell-mediated immune responses, including cytotoxic T-lymphocyte (CTL) reactions (Brazolot Milian, C. L, Weeratna, R., Krieg, A. M., Siegrist, C. A. & Davis, H. L. Proceedings of the National Academy of Sciences of the United States of America 95, 15553-15558 (1998).; Chu, R. S., Targoni, O. S., Krieg, A. M., Lehmann, P. V. & Harding, C. V. The Journal of experimental medicine 186, 1623-1631 (1997)). In this regard, CpG ODN has been considered a potential immunotherapeutic agent against inflammation, cancer, asthma, and hay fever (Krieg, A. M. Nature reviews. Drug discovery 5, 471-484 (2006); Klinman, D. M. Nature reviews. Immunology 4, 249-258 (2004)). A CpG oligodeoxynucleotide (CpG ODN) is a synthetic single stranded DNA comprising a non-methylated CpG motif with a immunostimulatory feature due to similarity with a microbial genome, and is recognized by TLR9 in a specific type of natural immune cell [Hartmann et al., J. Immunol. (2000) 164: 944-953; Wagner et al., Trends Immunol. (2004) 25: 1-6]. In ligand binding, TLR9 signals through an adapter molecule myD88 to induce the production of IRF7 dependent type I IFN and NF-κB dependent cytokines [Krieg et al., Nat. Rev. Drug Discov. (2006) 5: 471-84] Furthermore, it is reported that CpG ODN induces a Th1 response due to the type of cytokine induced by CpG ODN in APC in vivo [Krieg et al., Nat. Rev. Drug Discov. (2006) 5: 471-84]. Among different types of CpG ODN, type D CpG ODN strongly induces both type I and type II IFN, but cannot induce B cell activation [Krieg et al., Nat. Rev. Drug Discov. (2006) 5: 471-84; Klinman et al., Nat. Rev. Immunol. (2004) 4:1-10]. Type K CpG ODN (K3 CpG) strongly induces B cell activation to induce IL-6 and antibody production, but they only weakly induce type I and type II IFN. However, type D CpG ODN forms an aggregation, such that only type K CpG can be used for clinical applications [Krieg et al., Nat. Rev. Drug Discov. (2006) 5: 471-84; Klinman et al., Nat. Rev. Immunol. (2004) 4: 1-10].
Pathogen derived agents such as LPS or non-methylated CpG DNA (CpG) (CpG ODN) stimulate natural immune cells that produce cytokines such as type I or type II IFN and IL-12. This is useful in inducing a Th1 response and cell-mediated immunity [Kawai et al., Immunity. (2011) 34: 637-650; Trinchieri et al., Immunol. (2007) 7: 179-190]. IL-12 acts on naïve CD4+ T cells to derive the generation of Th1 and the production of IFNγ [Seder et al., Proc. Natl. Acad. Sci. U.S.A. (1993) 90: 10188-92; Hsieh et al., Science. (1993) 260: 547-579]. In addition, IFNγ producing Th1 cells are the main actors in the induction of type 1 immunity, which are distinguished by high phagocytic activity [Spellberg et al., Clin. Infect. Dis. (2001) 90509: 76-102; Mantovani et al., Curr. Opin. Immunol. (2010) 22: 231-237]. Furthermore, Th1 cells play an important role in the generation of antitumor immunity and are useful in CTL effector functions and suitable activation including IFNγ production [Hung et al., J. Exp. Med. (1998) 188: 2357-68; Vesely et al., Annu. Rev. Immunol. (2011) 29: 235-271]. Thus, agents, CTLs, and NK cells that can induce a strong Th1 response [Vitale et al., Eur. J. Immunol. (2014) 44: 1582-1592] may play an important role in the development of a vaccine adjuvant or immunotherapeutic agent that is effective against intracellular pathogens or cancer. Therefore, they are in immediate demand. Depending on the difference in backbone modification or surrounding sequences, they are classified into type D/A, type K/B, type C, and type P (Vollmer, J. & Krieg, A. M. Advanced drug delivery reviews 61, 195-204 (2009).) It is suggested that type D/A induces the production of type I interferon mainly from plasmacytoid dendritic cells (called “plasmacytoid DC” or “pDC”), and type K/B induces B cell growth and the production of IgM, IL-6 or the like. Type D/A CpG-DNA strongly induces IFN-α production, but exhibits low pDC maturation inducing activity and no direct immunostimulatory activity to B cells. Type K/B exhibits immunostimulatory activity to B cells, strongly promotes maturation of
pDCs, and has high IL-12 inducing capability, but has low IFN-α inducing capability. In type C sequences having repetitive sequences of TCG that are completely thiolated, IFN-α production by pDCs or polyclonal B cell activation is induced. Type D/A CpG ODN (also called type A, type D or the like and denoted as CpG-A ODN) is an oligonucleotide characterized by a phoshothioate (PS) bond at the 5′ and 3′ terminuses and by a poly G motif with a palindrom (palindromic structure) CpG containing sequence of phosphodiester (PO) in the middle. Cell uptake is facilitated due to the presence of phosphorothioate (PS) at the 5′ and 3′ terminuses. CpG type D/A produces a large quantity of interferon α (IFN-α) in pDCs (different feature from CpG type K/B). A potent activation and interferon gamma production are induced thereby in NK cells and γδ T cells. However, B cells are not activated and pDCs are not matured (Krug, A., et al. European journal of immunology 31, 2154-2163 (2001).; and Verthelyi, D., et al. Journal of immunology 166, 2372-2377 (2001).) Three other types of ODN consist of a PS backbone. Type K/B CpG ODN is also called CpG-type B or CpG-type K. All type K/B CpG ODN with one or more CpG motifs without a poly G motif have a phosphorothioate (PS) backbone. Typically, type K/B CpG ODN contains multiple CpG motifs with a non-palindromic structure. Type K/B CpG has weak IFN-α inducing activity (produces nearly none), but is a very potent Th1 adjuvant and a potent B cell response stimulating agent which produces IL-6 and activates and matures pDCs (Verthelyi, D., et al. Journal of immunology 166, 2372-2377 (2001); and Hartmann, G. & Krieg, A. M. Journal of immunology 164, 944-953 (2000)). Type K/B CpGODN has a function of promoting the survival, activating, and maturing both monocyte derived dendritic cells and pDCs. Recently developed type C and type P CpG ODN comprise one and two palindromic structure CpG sequences, respectively. Both can activate B
cells, like type K CpG ODN, and activate pDCs, like type D CpG ODN. Meanwhile, type C CpG ODN more weakly induces IFN-α production relative to type P CpG ODN (Hartmann, G., et al. European journal of immunology 33, 1633-1641 (2003); Marshall, J. D., et al. Journal of leukocyte biology 73, 781-792 (2003).; and Samulowitz, U., et al. Oligonucleotides 20, 93-101 (2010)). Type D/K and type P CpG ODN are shown to form a higher order structure i.e., Hoogsteen base pair forming a four parallel strand structure called G-tetrads and Watson-Crick base pair between a cis palindromic structure site and a trans palindromic structure site, respectively, which are required for potent IFN-α production by pDCs (Samulowitz, U., et al. Oligonucleotides 20, 93-101 (2010).; Kerkmann, M., et al. The Journal of biological chemistry 280, 8086-8093 (2005).; and Klein, D. et al. Ultramicroscopy 110, 689-693 (2010)). Due to the higher order structure, only type K and type C CpG ODN are generally considered usable as immunotherapeutic agents and vaccine adjuvants for humans (Puig, M., et al. Nucleic acids research 34, 6488-6495 (2006); Bode, C., et al. Expert review of vaccines 10, 499-511 (2011); and McHutchison, J. G., et al. Hepatology 46, 1341-1349 (2007)). In contrast to type A CpG ODN, type C CpG ODN has a complete phosphorothioate (PS) backbone without a poly G motif, but comprises the type A palindromic sequence of CpG in combination with a stimulatory CpG motif. It is reported from an in vivo study that type C CpG ODN is a very potent Th1 adjuvant. Type K CpG ODN used in a preferred embodiment in the present invention has a length of 10 nucleotides or longer and comprises the nucleotide sequence set forth in the following formula: 5′-N1N2N3T-CpG-WN4N5N6-3′ [Formula 1]
wherein the middle CpG motif (described as CpG) is not methylated, W is A or T, and N1, N2, N3, N4, N5, and N6 may be any nucleotide. In one embodiment, type K CpG ODN of the invention has a length of 10 nucleotides or longer and comprises the nucleotide sequence of the above-described formula. However, in the above-described formula, the CpG motif of 4 bases in the middle (TCpGW) only needs to be included in the 10 nucleotides. The motif does not necessarily need to be positioned between N3 and N4 in the above-described formula. Further, the N1, N2, N3, N4, N5, and N6 may be any nucleotide in the above-described formula. Combinations of at least one (preferably one) of N1 and N2, N2 and N3, N3 and N4, N4 and N5, and N5 and N6 may be a two base CpG motif. When the four base CpG motif is not positioned between N3 and N4, any two contiguous bases in the middle 4 bases (4th to 7th bases) in the above- described formula may be a CpG motif and the other two bases may be any nucleotide. Further, a part of or the entire phosphodiester bond of an oligodeoxynucleotide may be substituted with a phosphorothioate bond. Preferably, the entire phosphodiester bond of an oligodeoxynucleotide is substituted with a phosphorothioate bond. Type K CpG ODN suitably used in the present invention contains a non-palindromic structure comprising one or more CpG motifs. Type K CpG ODN more suitably used in the present invention consists of a non- palindromic structure comprising 1 or more CpG motifs. Type K CpG ODN contained in the oligodeoxynucleotide of the invention is preferably humanized. “Humanized” refers to having agonistic activity against human TLR9. Thus, the oligodeoxynucleotide of the invention comprising humanized type K CpG ODN has immunostimulatory activity unique to type K CpG ODN against humans (e.g., activity to activate human B cells to produce IL-6).
Humanized type K CpG ODN is generally characterized by a four base CpG motif consisting of TCGA or TCGT. In many cases, a single humanized type K CpG ODN comprises 2 or 3 of the four base CpG motifs. Thus, in a preferred embodiment, type K CpG ODN contained in the oligodeoxynucleotide of the invention comprises at least 1, more preferably 2 or more, and still more preferably 2 or 3 four base CpG motifs consisting of TCGA or TCGT. When such type K CpG ODN has 2 or 3 four base CpG motifs, these four base CpG motifs may be the same or different. However, this is not particularly limited, as long as there is agonist activity against human TLR9. One preferred type K CpG ODN included in the aspects of the invention comprises the nucleotide sequence set forth in the sequence (atcgactctc gagcgttctc (SEQ ID NO: 1)). Other suitable CpG ODNs include CpG 1826 (5′- tccatgacgttcctgacgtt-3′ (SEQ ID NO: 2)), D35 CpG (5′-ggtgcatcgatgcagggggg- 3′ (SEQ ID NO: 3)), and the like. One particularly preferred type K CpG ODN included in the aspects of the invention comprises the nucleotide sequence set forth in the sequence (atcgactctc gagcgttctc (SEQ ID NO:1)). Most preferably, the CpG ODN consists of SEQ ID NO:1 and is herein referred to as K3 CpG. The length of type K CpG ODN is not particularly limited, as long as the oligodeoxynucleotide of the invention activates immunostimulatory activity (e.g., activity to activate B cells (preferably human B cells) to produce IL-6) or has anticancer activity, but the length is preferably 100 nucleotides long or less (e.g., 10 to 75 nucleotides long). The length of type K CpG ODN is more preferably 50 nucleotides long or less (e.g., 10 to 40 nucleotides long). The length of type K CpG ODN is still more preferably 30 nucleotides long or less (e.g., 10 to 25 nucleotides long). The length of type K CpG ODN is most preferably 12 to 25 nucleotides long.
“STING” ((adapter molecule) stimulator of interferon genes)) identified as a membrane protein localized in the endoplasmic reticulum plays an important role in the biological defense mechanism against infections of various RNA viruses and DNA viruses. It is also reported that STING plays an important role in inducing natural immune responses against DNA components derived from microbes and viruses, but the molecular mechanism thereof had not been elucidated. STING can form a complex with not only genomic DNA derived from viruses, but also synthetic double stranded DNA of 45 to 90 base pairs called ISD and self-DNA components derived from apoptotic cells. Analysis of DNA interaction region in vitro demonstrated that the C-terminus side region of STING is important. Recognition of various DNA components by STING was demonstrated to induce dynamic local change to regions surrounding the nuclear membrane of STING and to induce interferon production via activation of TBK1. It is also suggested that STING is possibly involved in the regulation of chronic inflammatory responses via recognition of not only allo-DNA component from a microorganism, but also auto-DNA component. As used herein, a “STING ligand” and “STING agonist” are interchangeably used, which is a ligand (agonist) of “STING” ((adapter molecule) stimulator of interferon genes)) inducing type I IFN production and NF-κB mediated cytokine production. STING agonists are considered to be membrane proteins localized in the endoplasmic reticulum. As STING agonists, in addition to cGAMP, cyclic dinucleotides of microbial origin, c-di- AMP and c-di-GMP, are ligands of adapter molecule stimulators of IFN genes (STING), which signal through the TBK1-IRF3 axis to induce type I IFN production and NF-κB mediated cytokine production [Burdette et al., Nature. (2011) 478: 515-8; Mcwhirter et al., J. Exp. Med. (2009) 206: 1899- 1911]. Recent studies report that these cyclic dinucleotides function as a potent vaccine adjuvant due to their ability to enhance antigen-specific T cells and humoral immune responses. Despite the above, the inventors'
group has previously found that a STING agonist, DMXAA, unexpectedly induces a type 2 immune response via STING-IRF3 mediated type I IFN production [Tang et al., PLoS One. (2013) 8: 1-6]. Since type 2 immune responses can inhibit a type 1 immune response, the clinical usefulness of STING agonists, including cyclic dinucleotides, was debatable. For instance, the most common adjuvant, aluminum salt (alum), lacks the ability to induce cell-mediated immunity, which is understood to protect against cancer or diseases from intracellular pathogens [Hogenesch et al., Front. Immunol. (2013) 3: 1-13]. To overcome this limitation, alums were combined with many different types of adjuvants including monophosphoryl lipid A [Macleod et al., Proc. Natl. Acad. Sci. U.S.A (2011) 108: 7914-7919] and CpG ODN [Weeratna et al., Vaccine. (2000) 18: 1755-1762]. In regard to the techniques related to STING, especially when host DNA is unsuitably present in cytosol, host DNA may also be a sign of danger as in microorganism DNA, which results in interferon and inflammatory cytokine production [Desmet et al., Nat. Rev. Immunol. (2012) 12: 479-491; Barber et al., Immunol. Rev. (2011) 243: 99-108]. A recently identified cytosol DNA sensor is a cyclic GMP-AMP synthase (cGAS), which catalyzes the production of nonstandard cyclic dinucleotide cGAMP (2′3′-cGAMP) and contains a nonstandard 2′,5′ bond and 3′,5′ bond with the purine nucleoside thereof [Sun et al., Science. (2013) 339: 786-91]. Standard cGAMP (3′3′) is synthesized in a microbe and has more variety of bonds than mammalian 2′3′-cGAMP. GMP and AMP nucleosides bind by a bis-(3′,5′) bond [Wu et al., Science. (2013) 339: 826-30; Zhang et al., Mol. Cell. (2013) 51: 226-35]. Thus, examples of STING agonists that can be used in the present invention include cyclic dinucleotides (CDN) such as 2′3′-cGAMP, c-di-AMP, 3′3′-cGAMP, and 3′2′-cGAMP, xanthenone derivatives such as DMXAA, and the like. STING agonists are also explained in WO 2010/017248, whose entire content is incorporated herein by reference.
In preferred embodiments of the present invention, the STING agonists is c-di-AMP. Cyclic di-adenosine monophosphate (c-di-AMP) is a second messenger used in signal transduction in bacteria. As used herein, an “adjuvant” refers to an immunopotentiator that is added to increase the effect of a vaccine, which is an agent that is not a constituent of a specific antigen but increases immune responses to the administered antigen. The term “combination” as used herein is to be understood as referring to simultaneous, separate or sequential administration. In one aspect of the invention “combination” refers to simultaneous administration. In another aspect of the invention “combination” refers to separate administration. In a further aspect of the invention “combination” refers to sequential administration. Where the administration is sequential or separate, the delay in administering the second component is preferably such that both agents are present in the body so as to produce the effect of the combination. Hence, in aspects of this invention, a combination of two agents (a vaccine and an ICI; a vaccine and an adjuvant; a combination of two agents forming an adjuvant (CpG oligonucleotide and STING agonist) may be administered concomitantly, at different times, as part of the same formulation, as a combination of different formulations, in order, or separately. The term “allergy”, as used herein, refers to excessive immune responses to a specific antigen. Antigens from the environment causing allergies are especially called allergens. An “allergic disease” refers to a disease induced by an immune response to an exogenous antigen. However, this antigen is often harmless in a quantity that a patient is exposed to in normal life (e.g., pollen during spring time does not have toxicity in and of itself). An immune response resulting in unnecessary discomfort is experienced therewith. This is also called an allergic disease. Examples of typical diseases include atopic dermatitis, allergic rhinitis (hay fever),
allergic conjunctivitis, allergic gastroenteritis, bronchial asthma, childhood asthma, food allergy, drug allergy, and hives. Recently, pathological conditions exhibiting a type 1 allergy symptom such as asthma or facial flash only from the scent of citrus or fragrance of gum or the like has drawn attention. The term “N protein” refers to a viral nucleocapsid protein. The nucleic acid and proteins of each class of viruses assemble themselves into a structure called a nucleoprotein, or nucleocapsid. The nucleocapsid protein (N protein) is the most abundant protein in coronavirus. The N protein is a highly immunogenic phosphoprotein, and it is normally very conserved. The term N protein, in aspects of this invention, preferably refers to a coronavirus nucleocapsid protein, still more preferably to a SARS nucleocapsid (N) protein, and most preferably the SARS-CoV-2 N protein as displayed in Figure 2, or sequences having at least 70%, 80%, 90%, 95% or at least 99% sequence similarity with the sequence of Figure 2, determined over the entire length of the protein. Antiviral vaccination Cytotoxic T lymphocytes (CTLs) are key players in the immune control of cancer and (viral or bacterial) infection, as they recognize cancer or pathogen derived peptide epitopes presented by HLA class I molecules on the cancer cell or infected cell surface The antiviral vaccine strategy provided by the present invention targets the immune system against specific antigens in cancer. It achieves this by combining the viral antigen with a CpG oligonucleotide and a STING agonist as adjuvants. The present inventors are the first to show that this adjuvant combination can induce potent CTL responses in vivo. In particular, the combination of the adjuvants K3 CpG and c-di-AMP is preferred herein. The adjuvants and antigen may be used in solution, i.e. they do not need to be presented in the form of nanoparticles. Adjuvants and
antigen may be used in solution without adjuvants and antigen being conjugated to each other, nor peptides to be conjugated to carrier proteins. Adjuvants and antigen in solution do not need to be encapsulated in lipid or viral-like particles. In fact, the use of this adjuvant combination makes the use of nanoparticles, lipid particles, viral-like particles or conjugations to achieve cross-presentation and immunogenicity redundant. The use of the CpG oligonucleotide and a STING agonist combination, promotes cross- presentation while stimulating strong CTL immunity. This solves a major hurdle in viral peptide vaccination as it stimulates very strong CTL responses to peptides that otherwise need cross-presentation. The term “cross-presentation”, as used herein, refers to the process by which exogenous antigens captured by phagocytic antigen- presenting cells (APCs) are processed and presented onto MHC-I molecules. The vaccine compositions provided herein solves the problem of inducing cross-presentation without the need to generate modified or conjugated peptides or nanoparticles, lipid particles or viral-like particles in order to enhance uptake by APCs. Such modified peptides or nanoparticles would make it much more difficult to produce the vaccine composition under Good Manufacturing Practice (GMP) conditions, due to the chemicals required and complex production process. The presently provided vaccine composition requires only 3 components that can be mixed in aqueous solution, such as saline, and is easy to prepare and administer. These 3 components can be lyophilized together greatly facilitating the storage and transport of the vaccine without the need of a cold chain. Such a lyophilized vaccine can be reconstituted with sterile water just before usage. The adjuvant combination described in the present invention can be used with peptides such as synthetic long peptides (e.g., having a length of 10-30 consecutive amino acids) and solves the problem of immunogenicity when using peptides in vaccines. The inventors have shown this effect inter alia by using a 20 amino acid OVA peptide LEQLESIINFEKLTEWTSSN as
described in co-pending international application claiming priority from EP22386021.4. Peptide-based vaccines exhibit inefficient co-delivery of antigenic peptides and adjuvants to draining lymph nodes (dLNs), and can have the adverse effect of inducing immunological tolerance and reduced CD8+ T cell immunity. To solve this problem typical peptide vaccination protocols, conjugate peptides to a carrier protein or TLR agonists or present peptides in a multimeric format for co-delivery with adjuvant (such multimeric formats include virus-like-particles, nanoparticle or liposomes). The adjuvant combination described in the present invention can be used with peptides in solution to induce strong T cell immunity therefore avoiding the need for conjugations and multimeric particles. This is an important advantage as it simplifies manufacturing and safety concerns associated with conjugated molecules and multimeric particles. The vaccine compositions of the present invention overcome this problem and allow the use of such relatively long peptides, similarly without exact knowledge of the 8-10 amino acid sequence that is loaded in the MHC- class I complex, whereby efficient cross-presentation of these longer peptides is achieved by using a CpG oligonucleotide and a STING agonist as adjuvants, preferably, the combination of the adjuvants K3 CpG and c-di- AMP. In some vaccines K3 CpG may be substituted with humanized K3 CpG. Adjuvant combination One highly preferred adjuvant combination of a CpG oligonucleotide and a STING agonist as adjuvants is K3 CpG and c-di-AMP. This adjuvant combination is one aspect of this invention. The adjuvant combination may very suitably be used in combination with cancer neoantigens in (a vaccine for use in) the treatment of cancer. In embodiments, the invention provides the use of a combination
of (humanized) K3 CpG oligonucleotide and c-di-AMP as adjuvants together with a short peptide 10-30 amino acid long as antigen. These peptides can be derived from tumor neoantigens. The adjuvant combination may very suitably be used in combination with allergen immunotherapy, also known as desensitization or hypo-sensitization. Allergen-specific Immunotherapy (AIT) is the only available treatment aimed to tackle the underlying causes of allergy. The active components of subcutaneous vaccines traditionally consist of natural or modified allergen extracts which can be combined with adjuvant platforms. In aspects of this invention the adjuvant platform comprising the combination of K3 CpG and c-di-AMP is provided. The adjuvant combination may very suitably be used in combination with antigenic peptides from pathogens in the preparation of a vaccine that can be used to stimulate immunity against viruses, bacteria etc. Antiviral vaccine compositions The present invention provides antiviral vaccine compositions comprising a viral coat, matrix or core/capsid (glyco)protein as antigen, and further comprising as adjuvants a CpG oligonucleotide and a STING agonist. Preferably the CpG oligonucleotide is K3-CpG and the STING agonist is c-di-AMP. The present inventors have found that splenocytes of animals vaccinated with a vaccine comprising a SARS-CoV-2 nucleocapsid (N) protein as antigen and the K3 CpG + c-di-AMP combination as adjuvant, induced very strong IFNγ CD4 and CD8 T cell responses when restimulated with peptides that cover the full length N protein but no Th2 T cell responses. These Th1 and cytotoxic CD8 T responses are considered protective while Th2 responses are considered detrimental (See Examples).
The antiviral vaccine composition herein provided may also provide protection or treatment of other viral infectious diseases such as influenza (flu) caused by influenza viruses. The adjuvant combination of K3 CpG + c-di-AMP may be applied to viral vaccines in general, using any viral coat, matrix or core/capsid (glyco)protein as the antigen. The present invention further provides an antiviral vaccine composition comprising the adjuvant combination of K3 CpG + c-di-AMP, wherein the antigen is a SARS nucleocapsid (N) protein, preferably SARS- CoV-2 N protein. An antiviral vaccine composition in aspects of the invention comprises a viral coat, matrix or core/capsid (glyco)protein as antigen, and further comprising as adjuvants a CpG oligonucleotide and a STING agonist, preferably the antigen is a viral nucleocapsid protein, more preferably a SARS nucleocapsid (N) protein, even more preferably a SARS- CoV-2 N protein. A highly preferred embodiment of the CpG oligonucleotide and a STING agonist combination is in aspects of this invention is the combination K3 CpG + c-di-AMP. The present invention also provides a method of treating or preventing a viral infection, preferably a SARS infection, more preferably SARS-CoV-2 infection, comprising administrating to a subject in need thereof a therapeutically or prophylactically effective amount of a vaccine composition as described above comprising a viral coat, matrix or core/capsid (glyco)protein as the antigen. Medicaments and Dosage Forms The present invention is provided as medicaments (therapeutic agent or prophylactic agent) in various forms described above. The route of administration of a therapeutic agent, prophylactic agent, or the like that is effective upon therapy is preferably used, such as intravenous, subcutaneous, intramuscular, intraperitoneal, oral
administration, or the like. Examples of dosage form include injection, capsules, tablets, granules, and the like. The components of the present invention are effectively used upon administration as an injection. Aqueous solutions for injection may be stored, for example, in a vial or a stainless steel container. Aqueous solutions for injections may also be blended with, for example, saline, sugar (e.g., trehalose), NaCl, NaOH, or the like. Therapeutic agents may also be blended, for example, with a buffer (e.g., phosphate buffer), stabilizer, or the like. In general, the composition, medicament, therapeutic agent, prophylactic agent, or the like of the present invention comprises a therapeutically effective amount of a therapeutic agent or effective ingredient, and a pharmaceutically acceptable carrier or excipient. As used herein, “pharmaceutically acceptable” means that a substance is approved by a government regulatory agency or listed in the pharmacopoeia or other commonly recognized pharmacopoeia for use in animals, more specifically in humans. As used herein “carrier” refers to a diluent, adjuvant, excipient or vehicle administered with a therapeutic agent. Such a carrier can be an aseptic liquid such as water or oil, including, but not limited to, those derived from petroleum, animal, plant or synthesis, as well as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When a medicament is orally administered, water is a preferred carrier. For intravenous administration of a pharmaceutical composition, saline and aqueous dextrose are preferred carriers. Preferably, an aqueous saline solution and aqueous dextrose and glycerol solution are used as a liquid carrier of an injectable solution. Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, powdered skim milk, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal
diethylamino acetate, polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, saccharose, carboxymethylcellulose, corn starch, inorganic salt, and the like. When desirable, the composition can also contain a small amount of wetting agent, emulsifier, or pH buffer. These compositions can be in a form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release preparation, or the like. It is also possible to use traditional binding agents and carriers, such as triglyceride, to prepare a composition as a suppository. Oral preparation can also comprise a standard carrier such as medicine grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, or magnesium carbonate. Examples of a suitable carrier are described in E. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A.). Such a composition contains a therapeutically effective amount of therapy agent, preferably in a purified form, together with a suitable amount of carrier, such that the composition is provided in a form suitable for administration to a patient. A preparation must be suitable for the administration format. In addition, the composition may comprise, for example, a surfactant, excipient, coloring agent, flavoring agent, preservative, stabilizer, buffer, suspension, isotonizing agent, binding agent, disintegrant, lubricant, fluidity improving agent, corrigent, or the like. Examples of “salt” in one embodiment of the present invention include anionic salts formed with any acidic (e.g., carboxyl) group and cationic salts formed with any basic (e.g., amino) group. Salts include inorganic salts and organic salts, as well as salts described in, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19. Examples thereof further include metal salts, ammonium salts, salts with organic base, salts with inorganic acid, salts with organic acid, and the like. “Solvate” in one embodiment of the present invention is a compound formed with a solute or solvent. For example, J Honig et al., The Van Nostrand Chemist's Dictionary P650
(1953) can be referred for solvates. When a solvent is water, a solvate formed thereof is a hydrate. It is preferable that the solvent does not obstruct the biological activity of the solute. Examples of such a preferred solvent include, but not particularly limited to, water and various buffers. Examples of “chemical modification” in one embodiment of the present invention include modifications with PEG or a derivative thereof, fluorescein modification, biotin modification, and the like. When the present invention is administered as a medicament, various delivery systems are known, which can be used to administer the agent of the invention to a suitable site (e.g., esophagus). Examples of such a system include use of a recombinant cell that can express encapsulated therapeutic agent (e.g., polypeptide) in liposomes, microparticles, and microcapsules; use of endocytosis mediated by a receptor; construction of a therapy nucleic acid as a part of a retrovirus vector or another vector; and the like. Examples of the method of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. A medicament can be administered by any suitable route, such as by injection, bolus injection, or by absorption through epithelial or mucocutaneous lining (e.g., oral cavity, rectum, intestinal mucosa, or the like). In addition, an inhaler or mistifier using an aerosolizing agent can be used as needed. Moreover, other biological activating agents can also be administered concomitantly. Administration can be systemic or local. When the present invention is used for cancer, the present invention can be administered by any suitable route such as direct injection into cancer (lesion). In a preferred embodiment, a composition can be prepared as a pharmaceutical composition adapted to administration to humans in accordance with a known method. Such a composition can be administered by an injection. A composition for injection is typically a solution in an aseptic isotonic aqueous buffer. A composition can also comprise a local
anesthetic such as lidocaine, which alleviates the pain at the site of injection, and a solubilizing agent as needed. Generally, ingredients can be supplied individually or by mixing the ingredients together in a unit dosage form; and supplied, for example, in a sealed container such as an ampoule or sachet showing the amount of active agent or as a lyophilized powder or water-free concentrate. When a composition is to be administered by injection, the composition can be distributed using an injection bottle containing aseptic agent-grade water or saline. When composition is to be administered by injection, an aseptic water or saline ampoule for injection can also be provided such that the ingredients can be mixed prior to administration. The composition, medicament, therapeutic agent, and prophylactic agent of the invention can be prepared as a neutral or base form or other prodrugs (e.g., ester or the like). Pharmaceutically acceptable salts include salts formed with a free carboxyl group, derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, or the like, salts formed with a free amine group, derived from isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, or the like; and salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide or the like. The amount of therapeutic agent of the invention that is effective in therapy of a specific disorder or condition may vary depending on the properties of the disorder or condition. However, such an amount can be determined by those skilled in the art with a standard clinical technique based on the descriptions herein. Furthermore, an in vitro assay can be used in some cases to assist the identification of the optimal dosing range. The precise dose to be used for a preparation may also vary depending on the route of administration or the severity of the disease or disorder. Thus, the dose should be determined in accordance with the judgment of the attending physician or the condition of each patient. The dosage is not particularly
limited, but may be 0.001, 1, 5, 10, 15, 100, or 1000 mg/kg body weight per dosage or within a range between any two values described above. The dosing interval is not particularly limited, but may be, for example, 1 or 2 doses every 1, 7, 14, 21, or 28 days or 1 or 2 doses in a range of period between any two values described above. The dosage, dosing interval, and dosing method may be appropriately selected depending on the age, weight, symptom, target organ, or the like of the patient. Further, it is preferable that a therapeutic agent contains a therapeutically effective amount of effective ingredients, or an amount of effective ingredients effective for exerting a desired effect. When a malignant tumor marker significantly decreases after administration, the presence of a therapeutic effect may be acknowledged. The effective dose can be estimated from a dose-response curves obtained from in vitro or animal model testing systems. For SARS-CoV-2 N protein vaccination, the amount of N protein per vaccine injection may be in the range of about 100µg to 10mg, preferably about 500 µg to 5mg, or more preferably about 1mg to 3mg, or about 2mg. The amount of each adjuvant in the adjuvant combination comprised in a single dose vaccine injection is preferably about or equal in weight. The ratio between the ODN and STING agonist in an adjuvant combination of the invention may be 1:5 or 5:1, preferably the weight ratio is 1:2 to 2:1. Most preferably, the weight ratio is about 1:1. The amount of each adjuvant may be in the range of 100µg to 3mg. Hence, in preferred embodiments, an amount of K3 CpG of 100µg, 500 µg, 1mg, 2mg or 3mg may suitably be combined with an amount of c-di-AMP of 100µg, 500 µg, 1mg, 2mg or 3mg per vaccine injection. “Patient” or “subject” in one embodiment of the present invention includes humans and mammals excluding humans (e.g., one or more species of mice, guinea pigs, hamsters, rats, rabbits, pigs, sheep, goats, cows, horses, cats, dogs, marmosets, monkeys, and the like).
The pharmaceutical composition, therapeutic agent, or prophylactic agent of the invention can be provided as a kit. In a specific embodiment, the present invention provides an agent pack or kit comprising one or more containers filled with one or more ingredients of the composition or medicament of the invention. Optionally, information indicating approval for manufacture, use, or sale for administration to a human by a government agency regulating the manufacture, use, or sale of medicaments or biological products can be appended to such a container in a stipulated form. In a specific embodiment, the pharmaceutical composition comprising an ingredient of the present invention can be administered via liposomes, microparticles, or microcapsules. In various embodiments of the present invention, it may be useful to use such a composition to achieve sustained release of the ingredient of the present invention. The formulation procedure for the therapeutic agent, prophylactic agent, or the like of the invention as a medicament or the like is known in the art. The procedure is described, for example, in the Japanese Pharmacopoeia, the United States Pharmacopeia, pharmacopeia of other countries, or the like. Thus, those skilled in the art can determine the embodiment such as the amount to be used without undue experimentation from the descriptions herein. For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the disclosure includes embodiments having combinations of all or some of the features described. The content of the documents referred to herein is incorporated by reference.
EXAMPLES EXAMPLE 1. SARS-CoV-2 N protein plus K3 CpG + c-di-AMP vaccination induces strong memory in both CD4+ and CD8+ T cells Vaccination with SARS-CoV-2 nucleocapsid (N) protein as antigen and the K3 CpG + c-di-AMP combination as adjuvant, induces potent CD4 and CD8 T cell memory responses. Mice were vaccinated 3 times with 2- week intervals and 30 days after last immunization they were euthanized and the immune response against SARS-CoV-2 N protein was tested. Animals were vaccinated with 3µg of SARS-CoV-2 N protein and 10 µg of K3 CpG and 10 µg of c-di-AMP. For controls, the same amount of N protein was injected with AddaVax or 10 µg of c-di-AMP alone. Thirty days after last immunization, the time at which memory immunity is established, splenocytes were stimulated in vitro with peptide pools that cover the full SARS-CoV-2 N protein. Following 6h stimulation intracellular cytokines were stained and the number of cells producing IFNγ were measured by flow cytometry (Figure 5). PMA plus ionomycin was used as a positive control for the in vitro assay. Each group 5 animals. EXAMPLE 2. SARS CoV-2 N protein plus K3 CpG + c-di-AMP vaccination induces strong Th1 and CD8 T cell memory but no Th2 memory Vaccination with SARS-CoV-2 nucleocapsid (N) protein as antigen and the K3 CpG + c-di-AMP combination as adjuvant, induces potent IFNγ- producing Th1 CD4 and CD8 T cell memory responses with no Th2 (IL-5 producing) responses. Mice were vaccinated 3 times with 2-week intervals and 30 days after last immunization they were euthanized and the immune response against SARS-CoV-2 N protein was tested. Animals were vaccinated with 3µg of SARS-CoV-2 N protein and 10 µg of K3 CpG and 10 µg of c-di-AMP. Thirty days after last immunization, the time at which
memory immunity is established, splenocytes were stimulated in vitro in an ELISpot assay with peptide pools that cover the full SARS-CoV-2 N protein. IFNγ- or IL-5 producing T cells were measured by ELISpot assay. Each group 7-8 animals. Results are displayed in Table 1. Table 1. IFNγ- or IL-5 producing T cells as measured by ELISpot assay. IFN-γ SFC/1x106 splenocytes IL-5 SFC/1x106 splenocytes IFNγ Average IL-5 Average Unvaccinated 13.7 Unvaccinated 3.3 AddaVax 38.1 AddaVax 44.3 K3 CpG 26.7 K3 CpG 3.3 c-di-AMP 57.9 c-di-AMP 220.4 K3 CpG/c-di-AMP 768.6 K3 CpG/c-di-AMP 4.5 Materials & Methods to Examples 1 and 2 Animals. Eight to ten week-old female C57BL/6 mice were used for immunizations. To test the vaccine effect with or without anti-PD-1 treatment in tumor-bearing mice, 6-8 week-old female C57BL/6 mice were used. This study was carried out in accordance with the recommendations of the Instantie voor Dierenwelzijn (IvD), all studies were approved by the IvD (license AVD1010020209604). Specific pathogen free (SPF) C57BL/6 mice were purchased from Charles Rivers (Charles River Laboratories International, Inc. Wilmington, MA, USA) and housed in the Erasmus Dierenexperimenteel Center (EDC), Erasmus Medical Center animal facility (Rotterdam, The Netherlands), in groups of 2 to 4 mice and kept in IV cages, food and water was administered ad libitum. Proteins, peptides and adjuvants. Full length SARS-CoV-2 nucleocapsid protein (N protein; C- terminal His tagged full length protein of YP_009724397.2) was purchased
from GeneTex, Inc. Irvine, CA, USA. SARS-CoV-2 nucleoprotein peptide pool JPT PepMix SARS-CoV-2 (NCAP; 10215-mer peptides with 11 amino acid overlap) was purchased from JPT (JPT Peptide Technologies GmbH, Berlin, Germany). Adjuvants used were K3 CpG ((ATCG ACTC TCGA GCGT TCTC); GeneDesign Inc., Osaka, Japan) and c-di-AMP (CAS: 54447-84-6; Yamasa Corporation, Chiba, Japan). AddaVaxTM (InVivoGen, San Diego, USA) squalene-based commercial adjuvant was included as a control in some immunizations. SARS-CoV-2 nucleoprotein vaccinations. In order to test the SARS-CoV-2 nucleoprotein vaccine, mice were vaccinated subcutaneously in the left flank 3 times, once every two weeks. Thirty days after the last immunization mice were euthanized, and blood, spleen and left axillary and inguinal lymph nodes were collected for in vitro measurement of the memory T cell immune response. Mice were vaccinated with 100µl volume that contained 3µg of SARS-CoV-2 nucleoprotein, 10µg of K3 CpG and 10µg c-di-AMP adjuvants in sterile saline. For controls, the same amount of protein was injected in 100 µl sterile saline or 100 µl of a mix of equal volumes of sterile saline and AddaVaxTM. Flow cytometry. Single-cell suspensions of splenocytes and lymph nodes were generated by mechanical disruption of spleens and lymph nodes and then filtering through a 40µm cell strainer (Falcon, San Jose, CA, USA). Cells were washed and counted, 2x106 cells were used for staining. For blood staining 60µl of blood were used. After lysing erythrocytes, cells were washed and stained. In all stains cells were first pre-treated with Fc block for 10 min. For surface stains, cells were stained for 20 min on ice with different panels of antibodies. Additionally, cells were stained with PerCP- Cy 5.5 labeled Annexin V (BD Biosciences, San Jose, CA, USA) and PE
labeled-tetramers of H-2Kb MHC-I loaded with OVA(257-264). After staining, cells were washed with Hank's Balanced Salt Solution (HBSS) containing 3% fetal bovine serum (FBS) and 0.02% sodium azide, and fixed with 1% paraformaldehyde (PFA). When Annexin V was used all buffers contained 2.5mM CaCl2. For intracellular cytokine staining cells were stimulated with 10µg/ml SARS-CoV-2 nucleoprotein peptide pool for 6 hrs at 37°C in 5% CO2 in the presence of GolgiPlugTM (BD Biosciences) and CD107a-APC-Cy7 antibody. Cells were fixed overnight with IC Fixation Buffer at 4°C, washed with a Perm/Wash buffer (both from eBiosciences, Thermo Fisher Scientific, Waltham, MA, USA) and stained for intracellular cytokines for 45 min at 4°C. Fluorochrome conjugated antibodies for CD4 and CD8 were used. Finally, cells were washed twice with Perm/Wash buffer and fixed with 1% PFA. All samples were acquired in a Fortessa Flow Cytometer (BD Biosciences) and analyzed with FlowJo v.9.9.6 software. ELISPOT assay. The ELISPOT assays were performed according the manufacturer protocol (ImmunoSpot®, Cellular Technology Ltd (CTL), Shaker Heights, OH, USA). Ninety-six well ELISpot plates pre-coated overnight with anti- murine IFN-γ antibody or anti-murine IL-5 antibody according to manufacturer instructions. Plates were washed and 1x105 cells for IFN-γ plates or 4x105 cells for IL-5 plates were seeded in 200µl 5% FCS DMEM media per well. Cell were stimulated with media alone or SARS-CoV-2 nucleoprotein peptide pool (each peptide at 2µg/ml). As a positive control, PMA and Ionomycin were used at 20ng/ml and 500ng/ml final concentration respectively. Cells were incubated overnight at 37°C in 5% CO2. The next day the wells were washed twice with PBS and twice with PBS Tween 0.05%. Anti-murine detection antibody was added and incubated at room temperature for 2 hours. After washing 3 times the plates with PBS Tween
0.05%, Streptavidin solution was added and incubated at room temperature for 30min. Plates were then washed two more times with PBS Tween 0.05% and two times with deionized H2O and the developer solution was added and incubated at room temperature for 15min. The reaction was stopped by washing the plates with water, afterwards they were allowed to air-dry for at least 24 hrs before reading. Plates were read and analyzed using a CTL counter with ImmunoSpot Software (CTL, USA). EXAMPLE 3. K3 CpG plus c-di-AMP adjuvant combination when used with SARS-CoV-2 nucleocapsid protein (NC) elicits potent T cell immunity against SARS-CoV-2 NC K3 CpG plus c-di-AMP adjuvant combination when used with SARS-CoV-2 nucleocapsid protein (NC) elicits potent T cell immunity against SARS-CoV-2 NC (Figure 3). This vaccine retains its immunogenicity even when lyophilized by freeze drying (Figure 3C). Vaccination with SARS-CoV-2 NC together K3 CpG plus c-di-AMP adjuvant combination elicits strong memory Th1 immunity without memory Th2 immunity against NC protein which is elicited when c-di-AMP is used alone as an adjuvant (Figure 4A). Vaccination with SARS-CoV-2 NC together K3 CpG plus c-di-AMP adjuvant combination elicits CD4+ and CD8+ T cell memory (figure 2B). Lyophilized vaccine containing SARS-CoV-2 NC together with K3 CpG plus c-di-AMP adjuvant combination retains its activity (Figures 3 and 4). This is important as the vaccine does not require a cold chain for transport. To test protection against viral challenge with SARS-CoV-2 virus, animals were vaccinated as above with lyophilized vaccine and 28 days after the last boost animals were intranasally challenged with SARS-CoV-2 Wuhan strain (Figure 5).
Immunizations. SARS-CoV-2 nucleocapsid protein (NC) (GeneTex) was used for immunization. TLR9 agonist K type of CpG ODN (K3 CpG) and STING agonist c-di-AMP adjuvants were used in vivo at 10 µg per injection in sterile saline. K3 CpG was synthesized by GeneDesign (Japan) and c-di- AMP was kindly provided by Yamasa (Japan). AddavaxTM (InVivoGen, USA) was used by mixing it with an equal volume of sterile saline. In some experiments lyophilization of vaccines was tested. Vaccines containing SARS-CoV-2 nucleocapsid protein (NC) and K3 CpG + c-di-AMP were frozen in a -80°C freezer overnight and next day they were lyophilized by a Freeze- dryer Alpha 1-2 LDplus (CHRIST) overnight. To test the immunogenicity of the vaccines, C57BL/6 mice were immunized with 100 µl of saline, each vaccine containing (3 µg of) SARS- CoV-2 nucleocapsid protein (NC) and one of Addavax (3 µg of NC in Addavax), K3 CpG + c-di-AMP, or c-di-AMP, or lyophilized and water- reconstituted vaccine, containing 3 µg of NC and 10 µg K3 CpG + 10 µg c-di- AMP. Immunization occurred in the flank on days 0, 14, and 21. On days 21 and 35, blood were obtained for ELISpot analysis. On day 56, the mice were euthanized and blood and spleens were obtained for analysis. Flow cytometry. For flow cytometry measurement of NC-specific CD4+ and CD8+ T cells, single-cell suspensions of splenocytes, tissue were mechanically disrupted and filtered through a 40 µm cell strainer (Falcon, San Jose, CA, USA). Cells were washed and counted, and 2x106 cells were used for staining. For staining, cells were first pre-treated with anti-CD16/CD32 blocking antibodies for 10 min. For surface stains, cells were stained for 20 min on ice with surface staining antibodies. To exclude dead cells, cells were stained with PerCP-Cy 5.5-labeled Annexin V (BD Biosciences, San Jose, CA, USA) After staining, cells were washed with HBSS containing 3% FBS
and 0.02% sodium azide and fixed with 1% PFA. When Annexin V was used, all buffers contained 2.5 mM of CaCl2. For intracellular cytokine staining, cells were stimulated with 2 µg/ml PepMix SARS-CoV-2 (NCAP) (JPT, see above) for 18 hrs at 37°C in 5% CO2 in the presence of GolgiPlug (BD Bioscience) and anti-CD107a-APC-Cy7 antibody. Cells were surface stained as above including anti-CD107a-APC-Cy7, fixed ON with IC Fixation Buffer at 4°C, washed with a Perm/Wash buffer (eBiosciences) and stained for intracellular cytokines for 45 min at 4°C using fluorochrome conjugated antibodies. Finally, cells were washed twice with Perm/Wash buffer and fixed with 1% PFA. All samples were acquired in a Fortessa Flow Cytometer (BD Biosciences) and analyzed with FlowJo v.9.9.6 software. ELISPOT assay. For measuring NC-specific T cells by ELISPOT assay, plates pre- coated with anti-murine IFN-γ antibody or anti-murine IL-5 antibody were used (ImmunoSpot – CTL, USA). The protocol followed was as the manufacturer recommended. Briefly, 1x105 cells for IFN-γ plates and 4x105 cells for IL-5 plates were seeded in 100 µl per well, and 100 µl of media, PepMix SARS-CoV-2 (NCAP) (JPT) at 2 µg/ml were used to stimulate cells. Cells were incubated overnight at 37°C in 7% CO2. The next day the wells were washed twice with PBS and twice with PBS Tween 0.05%. Anti-murine detection antibody was added and incubated at RT for 2 hrs. After washing three times the plates with PBS Tween 0.05%, Streptavidin-AP solution was added and incubated at RT for 30 min. The plates were washed two more times with PBS Tween 0.05% and two times with deionized H2O and the developer solution was added and incubated at RT for 15 min. The reaction was stopped by washing the plates with water, afterwards they were allowed to air-dry for at least 24 hours before reading. The plates were read and analyzed in a CTL counter with ImmunoSpot Software (USA).
To test NC vaccine efficacy we vaccinated K18-hACE2 transgenic mice and then intranasally challenged with 104 PFU SARS-CoV2 Wuhan strain 50µl in DMEM. Mice were vaccinated SQ in the flank with lyophilized 3µg SARS-CoV-2 NC protein plus 10µg of K3 CpG and 10µg of c-di-AMP in 100 µl of sterile saline. Controls animals were be either injected with sterile saline alone or 3µg SARS-CoV-2 N protein in AddaVax. A negative control to test specificity of vaccine protection included lyophilized 3µg full length ovalbumin protein (OVA) plus 10µg of K3 CpG and 10µg of c-di-AMP in 100 µl of sterile saline. Mice were followed up to day 14 post viral challenge for weight loss and survival.
Claims
Claims 1. An antiviral vaccine composition, comprising a viral coat, matrix or core/capsid (glyco)protein as antigen, and an adjuvant combination of a CpG oligonucleotide and a STING agonist, optionally further comprising a pharmaceutically acceptable vehicle, preferably a non-particulate aqueous vehicle.
2. The antiviral vaccine composition according to claim 1, wherein the antigen is not a viral surface protein.
3. The antiviral vaccine composition according to claim 1, wherein the antigen is an internal viral protein.
4. The antiviral vaccine composition according to any one of claim 1- 3, wherein the viral coat, matrix or core/capsid (glyco)protein is from a seasonal virus, preferably selected from coronavirus, influenza virus, respiratory syncytial virus (RSV), norovirus, rhinovirus, parainfluenza, and metapneumovirus, more preferably coronavirus, influenza virus, and respiratory syncytial virus (RSV), even more preferably Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Influenza A and B virus.
5. The antiviral vaccine composition according to any one of claims 1-4, wherein the antigen is a SARS nucleocapsid (N) protein, preferably SARS-CoV-2 N protein.
6. The antiviral vaccine composition according to claim 4 or 5, wherein the virus is a virus that infects humans.
7. The antiviral vaccine composition according to any one of claims 1-6, wherein the CpG oligonucleotide is K3 CpG, and the STING agonist is c-di-AMP.
8. The antiviral vaccine composition according to any one of claims 1-7 in lyophilized form.
9. The antiviral vaccine composition according to any one of claims 1-8, for use in the prevention or treatment of viral infection.
10. A method of treating or preventing a viral infection, preferably a SARS infection, more preferably SARS-CoV-2 infection, in a subject in need thereof, comprising administering to said subject a therapeutically or prophylactically effective amount of an antiviral vaccine composition according to any one of claims 1-8.
11. The method of claim 10, wherein the CpG oligonucleotide is K3 CpG and the STING agonist is c-di-AMP.
12. An antiviral vaccine adjuvant combination comprising K3 CpG and c-di-AMP.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP22386021 | 2022-04-28 | ||
EP22386021.4 | 2022-04-28 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2023211281A1 true WO2023211281A1 (en) | 2023-11-02 |
Family
ID=81653535
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/NL2023/050230 WO2023211279A1 (en) | 2022-04-28 | 2023-04-28 | Adjuvant combinations for neopeptide vaccines |
PCT/NL2023/050232 WO2023211281A1 (en) | 2022-04-28 | 2023-04-28 | Antiviral vaccine composition |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/NL2023/050230 WO2023211279A1 (en) | 2022-04-28 | 2023-04-28 | Adjuvant combinations for neopeptide vaccines |
Country Status (1)
Country | Link |
---|---|
WO (2) | WO2023211279A1 (en) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010017248A2 (en) | 2008-08-04 | 2010-02-11 | University Of Miami | Sting (stimulator of interferon genes), a regulator of innate immune responses |
US11058758B2 (en) * | 2014-11-20 | 2021-07-13 | National Institutes Of Biomedical Innovation, Health And Nutrition | TH1-inducing adjuvant comprising combination of different nucleic acid adjuvants, and use of same |
KR20210123234A (en) * | 2020-04-02 | 2021-10-13 | 조선대학교산학협력단 | Recombinant nucleocapsid protein for diagnosis and vaccine of COVID-19 and use thereof |
-
2023
- 2023-04-28 WO PCT/NL2023/050230 patent/WO2023211279A1/en unknown
- 2023-04-28 WO PCT/NL2023/050232 patent/WO2023211281A1/en unknown
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010017248A2 (en) | 2008-08-04 | 2010-02-11 | University Of Miami | Sting (stimulator of interferon genes), a regulator of innate immune responses |
US11058758B2 (en) * | 2014-11-20 | 2021-07-13 | National Institutes Of Biomedical Innovation, Health And Nutrition | TH1-inducing adjuvant comprising combination of different nucleic acid adjuvants, and use of same |
KR20210123234A (en) * | 2020-04-02 | 2021-10-13 | 조선대학교산학협력단 | Recombinant nucleocapsid protein for diagnosis and vaccine of COVID-19 and use thereof |
Non-Patent Citations (51)
Title |
---|
AKONDY, R.S. ET AL., NATURE, vol. 552, no. 7685, 2017, pages 362 - 367 |
AKONDY, R.S. ET AL., PROC NATL ACAD SCI USA, vol. 112, 2015, pages 3050 - 3055 |
BARBER ET AL., IMMUNOL. REV., vol. 243, 2011, pages 99 - 108 |
BERGE ET AL., J. PHARM. SCI., vol. 66, 1977, pages 1 - 19 |
BODE, C. ET AL., EXPERT REVIEW OF VACCINES, vol. 10, 2011, pages 499 - 511 |
BRAZOLOT MILIAN, C. LWEERATNA, R.KRIEG, A. M.SIEGRIST, C. A.DAVIS, H. L., PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 95, 1998, pages 15553 - 15558 |
BURCU TEMIZOZ ET AL: "TLR9 and STING agonists synergistically induce innate and adaptive type-II IFN", EUROPEAN JOURNAL OF IMMUNOLOGY, WILEY-VCH, HOBOKEN, USA, vol. 45, no. 4, 5 February 2015 (2015-02-05), pages 1159 - 1169, XP071227632, ISSN: 0014-2980, DOI: 10.1002/EJI.201445132 * |
BURDETTE ET AL., NATURE, vol. 478, 2011, pages 515 - 8 |
CHU, R. S.TARGONI, O. S.KRIEG, A. M.LEHMANN, P. V.HARDING, C. V., THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 186, 1997, pages 1623 - 1631 |
DEL GIUDICE, G. ET AL., SEMIN IMMUNOL, vol. 39, 2018, pages 14 - 21 |
DESMET ET AL., NAT. REV. IMMUNOL., vol. 12, 2012, pages 479 - 491 |
DOROSTKAR FARIBA ET AL: "Co-administration of 2'3'-cGAMP STING activator and CpG-C adjuvants with a mutated form of HPV 16 E7 protein leads to tumor growth inhibition in the mouse model", INFECTIOUS AGENTS AND CANCER, vol. 16, no. 1, 1 December 2021 (2021-12-01), XP093066581, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836183/pdf/13027_2021_Article_346.pdf> DOI: 10.1186/s13027-021-00346-7 * |
HARTMANN ET AL., J. IMMUNOL., vol. 164, 2000, pages 944 - 953 |
HARTMANN, G. ET AL., EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 33, 2003, pages 1633 - 1641 |
HARTMANN, G.KRIEG, A. M., JOURNAL OF IMMUNOLOGY, vol. 164, 2000, pages 944 - 953 |
HEMMI, H. ET AL., NATURE, vol. 408, 2000, pages 740 - 745 |
HOGENESCH ET AL., FRONT. IMMUNOL., vol. 3, 2013, pages 1 - 13 |
HSIEH ET AL., SCIENCE, vol. 339, 2013, pages 826 - 579 |
HUNG ET AL., J. EXP. MED., vol. 188, 1998, pages 2357 - 68 |
J HONIG ET AL.: "The Van Nostrand Chemist's Dictionary P650", 1953 |
KAWAI ET AL., , IMMUNITY, vol. 34, 2011, pages 637 - 650 |
KERKMANN, M. ET AL., THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 280, 2005, pages 8086 - 8093 |
KLEIN, D. ET AL., ULTRAMICROSCOPY, vol. 110, 2010, pages 689 - 693 |
KLINMAN ET AL., NAT. REV. IMMUNOL., vol. 4, 2004, pages 1 - 10 |
KLINMAN, D. M., NATURE REVIEWS. IMMUNOLOGY, vol. 4, 2004, pages 249 - 258 |
KRIEG ET AL., NAT. REV. DRUG DISCOV., vol. 5, 2006, pages 471 - 84 |
KRIEG, A. M., NATURE REVIEWS. DRUG DISCOVERY, vol. 5, 2006, pages 471 - 484 |
KRUG, A. ET AL., EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 31, 2001, pages 2154 - 2163 |
MACLEOD ET AL., PROC. NATL. ACAD. SCI. U.S.A, vol. 108, 2011, pages 7914 - 7919 |
MANTOVANI ET AL., CURR. OPIN. IMMUNOL., vol. 22, 2010, pages 231 - 237 |
MARSHALL, J. D. ET AL., JOURNAL OF LEUKOCYTE BIOLOGY, vol. 73, 2003, pages 781 - 792 |
MCHUTCHISON, J. G. ET AL., HEPATOLOGY, vol. 46, 2007, pages 1341 - 1349 |
MCWHIRTER ET AL., J. EXP. MED., vol. 206, 2009, pages 1899 - 1911 |
MILLER, J.D. ET AL., IMMUNITY, vol. 28, 2008, pages 710 - 722 |
PUIG, M. ET AL., NUCLEIC ACIDS RESEARCH, vol. 34, 2006, pages 6488 - 6495 |
PULENDRAN, B. ET AL., NAT REV DRUG DISCOV, vol. 20, 2021, pages 454 - 475 |
RAPAKA REKHA R. ET AL: "Using Adjuvants to Drive T Cell Responses for Next-Generation Infectious Disease Vaccines", VACCINES, vol. 9, no. 8, 24 July 2021 (2021-07-24), pages 820, XP093067068, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8402546/pdf/vaccines-09-00820.pdf> DOI: 10.3390/vaccines9080820 * |
SAMULOWITZ, U. ET AL., OLIGONUCLEOTIDES, vol. 20, 2010, pages 93 - 101 |
SCHMIDT MEGAN E. ET AL: "The CD8 T Cell Response to Respiratory Virus Infections", vol. 9, 1 January 2018 (2018-01-01), pages 678, XP055968420, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5900024/pdf/fimmu-09-00678.pdf> DOI: 10.3389/fimmu.2018.00678 * |
SEDER ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 90, 1993, pages 10188 - 92 |
SPELLBERG ET AL., CLIN. INFECT. DIS., vol. 90509, 2001, pages 76 - 102 |
TANG ET AL., PLOS ONE, vol. 8, 2013, pages 1 - 6 |
TEMIZOZ BURCU ET AL: "Anti-tumor immunity by transcriptional synergy between TLR9 and STING activation", IMMUNOLOGY FRONTIER RESEARCH CENTER (IFREC), OSAKA UNIVERSITY , OSAKA , JAPAN, vol. 34, no. 7, 14 April 2022 (2022-04-14), pages 353 - 364, XP093066652, Retrieved from the Internet <URL:https://watermark.silverchair.com/dxac012.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAAA2gwggNkBgkqhkiG9w0BBwagggNVMIIDUQIBADCCA0oGCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMqfFdE-rEFjumWhHwAgEQgIIDG3zAzbLKnGRF_PuCCP3yT4v_NFvNRR9KzFdEeIsWhb_CIJ3y2Ofh3FFL0ml896j81p2zgIUcq1pn1pBaqGGgonKWEHnb> DOI: 10.1093/intimm/dxac012 * |
TRINCHIERI ET AL., , IMMUNOL., vol. 7, 2007, pages 179 - 190 |
VERTHELYI, D. ET AL., JOURNAL OF IMMUNOLOGY, vol. 166, 2001, pages 2372 - 2377 |
VESELY ET AL., ANNU. REV. IMMUNOL., vol. 29, 2011, pages 235 - 271 |
VITALE ET AL., EUR. J. IMMUNOL., vol. 44, 2014, pages 1582 - 1592 |
VOLLMER, J.KRIEG, A. M., ADVANCED DRUG DELIVERY REVIEWS, vol. 61, 2009, pages 195 - 204 |
WAGNER ET AL., TRENDS IMMUNOL, vol. 25, 2004, pages 1 - 6 |
WEERATNA ET AL., VACCINE., vol. 18, 2000, pages 1755 - 1762 |
ZHANG ET AL., MOL. CELL, vol. 51, 2013, pages 226 - 35 |
Also Published As
Publication number | Publication date |
---|---|
WO2023211279A1 (en) | 2023-11-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20230210965A1 (en) | PRIME-BOOST REGIMENS INVOLVING ADMINISTRATION OF AT LEAST ONE mRNA CONSTRUCT | |
JP6762030B2 (en) | New Th1-inducible adjuvants and their uses by combining different nucleic acid adjuvants | |
KR101569218B1 (en) | Emulsion vaccine compositions comprising antigen and adjuvant in the aqueous phase | |
EP3888676A1 (en) | Immunogenic combinations | |
AU2013204906B2 (en) | Adjuvant and vaccine compositions | |
JP2009523721A (en) | Immunogenic substances comprising an adjuvant based on polyinosinic acid-polycytidylic acid | |
JP2009523722A (en) | Mucosal immunogenic substances containing adjuvants based on polyinosinic acid-polycytidylic acid | |
WO2016199904A1 (en) | Adjuvant for vaccines, vaccine, and immunity induction method | |
WO2005121378A2 (en) | Methods and compositions for vaccination | |
Akache et al. | Adjuvants: Engineering protective immune responses in human and veterinary vaccines | |
WO2012074881A2 (en) | Vaccines against herpes simplex virus type 2: compositions and methods for eliciting an immune response | |
WO2023211281A1 (en) | Antiviral vaccine composition | |
CN117897487A (en) | Application of artificially synthesized CpG-containing single-chain deoxyoligonucleotide in vaccine | |
JP2003528818A (en) | Induction of mucosal immunity by vaccination via the skin route | |
US20120231044A1 (en) | Vaccine formulation of mannose coated peptide particles | |
Morein et al. | New ISCOMs meet unsettled vaccine demands | |
US20220257752A1 (en) | New use of cyclic dinucleotides | |
US20240148864A1 (en) | Polysaccharide adjuvants for virus vaccines | |
US20240115693A1 (en) | Sars-cov-2 antigen nanoparticles and uses there of | |
WO2023240278A2 (en) | Uses of glycolipids as a vaccine adjuvant and methods thereof | |
Liu et al. | Use of novel DNA vectors and immunologic adjuvants in HIV vaccine development | |
US10174100B1 (en) | Multivalent DNA composition for Yersinia pestis | |
Huber et al. | Whole-inactivated influenza virus as an adjuvant for influenza peptide antigens | |
Soema et al. | Whole inactvated influenza virus as an adjuvant for influenza peptide antigens | |
Pertmer | Characterization of immune responses following neonatal DNA immunization: a dissertation |
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: 23721790 Country of ref document: EP Kind code of ref document: A1 |