WO2012023995A1 - Modification d'une protéine de capside d'adénovirus recombinant par des épitopes immunogènes de protéine circumsporozoïte de plasmidium - Google Patents
Modification d'une protéine de capside d'adénovirus recombinant par des épitopes immunogènes de protéine circumsporozoïte de plasmidium Download PDFInfo
- Publication number
- WO2012023995A1 WO2012023995A1 PCT/US2011/025535 US2011025535W WO2012023995A1 WO 2012023995 A1 WO2012023995 A1 WO 2012023995A1 US 2011025535 W US2011025535 W US 2011025535W WO 2012023995 A1 WO2012023995 A1 WO 2012023995A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nanp
- adenovirus
- seq
- sequence
- hvr1
- Prior art date
Links
- 241000701161 unidentified adenovirus Species 0.000 title claims abstract description 240
- 108700005482 Protozoan circumsporozoite Proteins 0.000 title claims abstract description 41
- 230000002163 immunogen Effects 0.000 title claims abstract description 25
- 108090000565 Capsid Proteins Proteins 0.000 title claims description 38
- 102100023321 Ceruloplasmin Human genes 0.000 title claims description 20
- 230000004048 modification Effects 0.000 title description 7
- 238000012986 modification Methods 0.000 title description 7
- 101900205473 Plasmodium falciparum Circumsporozoite protein Proteins 0.000 claims abstract description 97
- NTIZESTWPVYFNL-UHFFFAOYSA-N Methyl isobutyl ketone Chemical class CC(C)CC(C)=O NTIZESTWPVYFNL-UHFFFAOYSA-N 0.000 claims abstract description 64
- 201000004792 malaria Diseases 0.000 claims abstract description 61
- 210000003719 b-lymphocyte Anatomy 0.000 claims abstract description 54
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 50
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 claims abstract description 34
- 239000013600 plasmid vector Substances 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 32
- 230000000890 antigenic effect Effects 0.000 claims abstract description 25
- 239000002773 nucleotide Substances 0.000 claims abstract description 10
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 10
- KQPKMEYBZUPZGK-UHFFFAOYSA-N 4-[(4-azido-2-nitroanilino)methyl]-5-(hydroxymethyl)-2-methylpyridin-3-ol Chemical compound CC1=NC=C(CO)C(CNC=2C(=CC(=CC=2)N=[N+]=[N-])[N+]([O-])=O)=C1O KQPKMEYBZUPZGK-UHFFFAOYSA-N 0.000 claims description 244
- 101001111984 Homo sapiens N-acylneuraminate-9-phosphatase Proteins 0.000 claims description 228
- 102100023906 N-acylneuraminate-9-phosphatase Human genes 0.000 claims description 228
- 101000666856 Homo sapiens Vasoactive intestinal polypeptide receptor 1 Proteins 0.000 claims description 68
- 102100038388 Vasoactive intestinal polypeptide receptor 1 Human genes 0.000 claims description 68
- 108090000623 proteins and genes Proteins 0.000 claims description 57
- 229960005486 vaccine Drugs 0.000 claims description 55
- 208000015181 infectious disease Diseases 0.000 claims description 40
- 239000013598 vector Substances 0.000 claims description 36
- 239000002671 adjuvant Substances 0.000 claims description 23
- 150000007523 nucleic acids Chemical group 0.000 claims description 22
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 12
- 230000003472 neutralizing effect Effects 0.000 claims description 12
- 230000028996 humoral immune response Effects 0.000 claims description 10
- 230000024932 T cell mediated immunity Effects 0.000 claims description 9
- 230000001939 inductive effect Effects 0.000 claims description 5
- 230000037452 priming Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 abstract description 33
- 230000036039 immunity Effects 0.000 abstract description 20
- 108700019146 Transgenes Proteins 0.000 abstract description 18
- 229940124735 malaria vaccine Drugs 0.000 abstract description 12
- 230000028993 immune response Effects 0.000 abstract description 10
- 230000003405 preventing effect Effects 0.000 abstract description 10
- 230000001225 therapeutic effect Effects 0.000 abstract description 9
- 230000003263 anti-adenoviral effect Effects 0.000 abstract description 6
- 230000009145 protein modification Effects 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 58
- 102000004169 proteins and genes Human genes 0.000 description 50
- 235000018102 proteins Nutrition 0.000 description 48
- 239000012634 fragment Substances 0.000 description 42
- 238000003752 polymerase chain reaction Methods 0.000 description 35
- 210000000234 capsid Anatomy 0.000 description 27
- 210000004027 cell Anatomy 0.000 description 26
- 241000223960 Plasmodium falciparum Species 0.000 description 25
- 150000001413 amino acids Chemical group 0.000 description 24
- 230000004083 survival effect Effects 0.000 description 19
- 101710132601 Capsid protein Proteins 0.000 description 18
- 244000045947 parasite Species 0.000 description 18
- 239000013615 primer Substances 0.000 description 18
- 241000700605 Viruses Species 0.000 description 17
- 239000004480 active ingredient Substances 0.000 description 17
- 230000009261 transgenic effect Effects 0.000 description 17
- 210000001744 T-lymphocyte Anatomy 0.000 description 16
- 102000039446 nucleic acids Human genes 0.000 description 15
- 108020004707 nucleic acids Proteins 0.000 description 15
- 108090000765 processed proteins & peptides Chemical group 0.000 description 14
- 210000003046 sporozoite Anatomy 0.000 description 13
- 241000282414 Homo sapiens Species 0.000 description 12
- 241000699670 Mus sp. Species 0.000 description 12
- 235000001014 amino acid Nutrition 0.000 description 12
- 239000000427 antigen Substances 0.000 description 12
- 108091007433 antigens Proteins 0.000 description 12
- 102000036639 antigens Human genes 0.000 description 12
- 239000003814 drug Substances 0.000 description 12
- 230000003053 immunization Effects 0.000 description 12
- 101710145505 Fiber protein Proteins 0.000 description 11
- 241000224016 Plasmodium Species 0.000 description 11
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 11
- 201000010099 disease Diseases 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 230000014509 gene expression Effects 0.000 description 11
- 238000002649 immunization Methods 0.000 description 11
- 239000002953 phosphate buffered saline Substances 0.000 description 11
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 230000001681 protective effect Effects 0.000 description 10
- 210000002845 virion Anatomy 0.000 description 10
- 238000009472 formulation Methods 0.000 description 9
- 230000008348 humoral response Effects 0.000 description 9
- 238000003780 insertion Methods 0.000 description 9
- 230000037431 insertion Effects 0.000 description 9
- 239000002245 particle Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 241001135569 Human adenovirus 5 Species 0.000 description 8
- 210000004369 blood Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 239000000835 fiber Substances 0.000 description 8
- 244000052769 pathogen Species 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 241000223830 Plasmodium yoelii Species 0.000 description 7
- 239000000654 additive Substances 0.000 description 7
- 230000001413 cellular effect Effects 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 108091033319 polynucleotide Proteins 0.000 description 7
- 102000040430 polynucleotide Human genes 0.000 description 7
- 239000002157 polynucleotide Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 101710117490 Circumsporozoite protein Proteins 0.000 description 6
- 101100082057 Mus musculus Aldh18a1 gene Proteins 0.000 description 6
- 229940021704 adenovirus vaccine Drugs 0.000 description 6
- 238000010586 diagram Methods 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 239000013022 formulation composition Substances 0.000 description 6
- 230000005847 immunogenicity Effects 0.000 description 6
- 230000004044 response Effects 0.000 description 6
- 241000282412 Homo Species 0.000 description 5
- 241000224017 Plasmodium berghei Species 0.000 description 5
- 230000005867 T cell response Effects 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000003937 drug carrier Substances 0.000 description 5
- 210000003743 erythrocyte Anatomy 0.000 description 5
- 230000004727 humoral immunity Effects 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 238000007912 intraperitoneal administration Methods 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 229920001184 polypeptide Polymers 0.000 description 5
- 230000003389 potentiating effect Effects 0.000 description 5
- 230000003449 preventive effect Effects 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 239000013605 shuttle vector Substances 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 230000003612 virological effect Effects 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 4
- 208000030852 Parasitic disease Diseases 0.000 description 4
- 241000223821 Plasmodium malariae Species 0.000 description 4
- 206010035501 Plasmodium malariae infection Diseases 0.000 description 4
- 241000223810 Plasmodium vivax Species 0.000 description 4
- 238000000137 annealing Methods 0.000 description 4
- 210000000612 antigen-presenting cell Anatomy 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 230000000875 corresponding effect Effects 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 210000002443 helper t lymphocyte Anatomy 0.000 description 4
- 230000002458 infectious effect Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 229930182490 saponin Natural products 0.000 description 4
- 235000017709 saponins Nutrition 0.000 description 4
- 150000007949 saponins Chemical class 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000007920 subcutaneous administration Methods 0.000 description 4
- 229940124597 therapeutic agent Drugs 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 238000013519 translation Methods 0.000 description 4
- 238000002255 vaccination Methods 0.000 description 4
- CXURGFRDGROIKG-UHFFFAOYSA-N 3,3-bis(chloromethyl)oxetane Chemical compound ClCC1(CCl)COC1 CXURGFRDGROIKG-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108700026758 Adenovirus hexon capsid Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 108010077850 Nuclear Localization Signals Proteins 0.000 description 3
- 241000224024 Plasmodium chabaudi Species 0.000 description 3
- 241000223801 Plasmodium knowlesi Species 0.000 description 3
- 241001505293 Plasmodium ovale Species 0.000 description 3
- 206010035502 Plasmodium ovale infection Diseases 0.000 description 3
- -1 QS21 ) Chemical class 0.000 description 3
- 241000283984 Rodentia Species 0.000 description 3
- 108020005202 Viral DNA Proteins 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000000987 immune system Anatomy 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 210000004962 mammalian cell Anatomy 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 210000003936 merozoite Anatomy 0.000 description 3
- 229940035032 monophosphoryl lipid a Drugs 0.000 description 3
- 230000036281 parasite infection Effects 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 239000013603 viral vector Substances 0.000 description 3
- QSLFDILMORXPKP-UHFFFAOYSA-N (3-methylimidazol-3-ium-1-yl)-methylsulfanylphosphinate Chemical compound CSP([O-])(=O)N1C=C[N+](C)=C1 QSLFDILMORXPKP-UHFFFAOYSA-N 0.000 description 2
- 101710203310 Apical membrane antigen 1 Proteins 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 102100036411 Dermatopontin Human genes 0.000 description 2
- 101710088341 Dermatopontin Proteins 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 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 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101710094396 Hexon protein Proteins 0.000 description 2
- 101710192606 Latent membrane protein 2 Proteins 0.000 description 2
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 description 2
- 241000714474 Rous sarcoma virus Species 0.000 description 2
- 101710097904 Serine-rich antigen Proteins 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229930006000 Sucrose Natural products 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
- 101710137302 Surface antigen S Proteins 0.000 description 2
- 101710109576 Terminal protein Proteins 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000078 anti-malarial effect Effects 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000020411 cell activation Effects 0.000 description 2
- 230000007969 cellular immunity Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 230000000120 cytopathologic effect Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 239000006196 drop Substances 0.000 description 2
- 230000012202 endocytosis Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 210000003494 hepatocyte Anatomy 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 239000000568 immunological adjuvant Substances 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 230000008488 polyadenylation Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 230000001568 sexual effect Effects 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 102000055046 tissue-factor-pathway inhibitor 2 Human genes 0.000 description 2
- 108010016054 tissue-factor-pathway inhibitor 2 Proteins 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 239000013638 trimer Substances 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-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
- 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 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- RHKWIGHJGOEUSM-UHFFFAOYSA-N 3h-imidazo[4,5-h]quinoline Chemical class C1=CN=C2C(N=CN3)=C3C=CC2=C1 RHKWIGHJGOEUSM-UHFFFAOYSA-N 0.000 description 1
- OPVPGKGADVGKTG-BQBZGAKWSA-N Ac-Asp-Glu Chemical group CC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(O)=O OPVPGKGADVGKTG-BQBZGAKWSA-N 0.000 description 1
- 108010042708 Acetylmuramyl-Alanyl-Isoglutamine Proteins 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000256186 Anopheles <genus> Species 0.000 description 1
- AXXCUABIFZPKPM-BQBZGAKWSA-N Asp-Arg-Gly Chemical group [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O AXXCUABIFZPKPM-BQBZGAKWSA-N 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 230000003844 B-cell-activation Effects 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 101710198223 Capsid fiber protein Proteins 0.000 description 1
- 102000001327 Chemokine CCL5 Human genes 0.000 description 1
- 108010055166 Chemokine CCL5 Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 108010035601 Coxsackie and Adenovirus Receptor Like Membrane Protein Proteins 0.000 description 1
- 102000008198 Coxsackie and Adenovirus Receptor Like Membrane Protein Human genes 0.000 description 1
- 241000709687 Coxsackievirus Species 0.000 description 1
- 108091029523 CpG island Proteins 0.000 description 1
- 241000938605 Crocodylia Species 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 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 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 241000255925 Diptera Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 206010015150 Erythema Diseases 0.000 description 1
- 108010040721 Flagellin Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 1
- 101000831496 Homo sapiens Toll-like receptor 3 Proteins 0.000 description 1
- 101000669447 Homo sapiens Toll-like receptor 4 Proteins 0.000 description 1
- 101000669460 Homo sapiens Toll-like receptor 5 Proteins 0.000 description 1
- 101000669402 Homo sapiens Toll-like receptor 7 Proteins 0.000 description 1
- 101000800483 Homo sapiens Toll-like receptor 8 Proteins 0.000 description 1
- 241000701149 Human adenovirus 1 Species 0.000 description 1
- 241000598171 Human adenovirus sp. Species 0.000 description 1
- 108010042653 IgA receptor Proteins 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
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 101000668058 Infectious salmon anemia virus (isolate Atlantic salmon/Norway/810/9/99) RNA-directed RNA polymerase catalytic subunit Proteins 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000013462 Interleukin-12 Human genes 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 108010028921 Lipopeptides Proteins 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 102000029749 Microtubule Human genes 0.000 description 1
- 108091022875 Microtubule Proteins 0.000 description 1
- 101710151805 Mitochondrial intermediate peptidase 1 Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 208000000112 Myalgia Diseases 0.000 description 1
- 108010047956 Nucleosomes Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 208000009182 Parasitemia Diseases 0.000 description 1
- 102000010292 Peptide Elongation Factor 1 Human genes 0.000 description 1
- 108010077524 Peptide Elongation Factor 1 Proteins 0.000 description 1
- 241001505483 Plasmodium falciparum 3D7 Species 0.000 description 1
- RVGRUAULSDPKGF-UHFFFAOYSA-N Poloxamer Chemical compound C1CO1.CC1CO1 RVGRUAULSDPKGF-UHFFFAOYSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 1
- 108010008038 Synthetic Vaccines Proteins 0.000 description 1
- 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 1
- 102000002938 Thrombospondin Human genes 0.000 description 1
- 108060008245 Thrombospondin Proteins 0.000 description 1
- 108010046075 Thymosin Proteins 0.000 description 1
- 102000007501 Thymosin Human genes 0.000 description 1
- 108010060818 Toll-Like Receptor 9 Proteins 0.000 description 1
- 102000002689 Toll-like receptor Human genes 0.000 description 1
- 108020000411 Toll-like receptor Proteins 0.000 description 1
- 102100024324 Toll-like receptor 3 Human genes 0.000 description 1
- 102100039360 Toll-like receptor 4 Human genes 0.000 description 1
- 102100039357 Toll-like receptor 5 Human genes 0.000 description 1
- 102100039390 Toll-like receptor 7 Human genes 0.000 description 1
- 102100033110 Toll-like receptor 8 Human genes 0.000 description 1
- 102100033117 Toll-like receptor 9 Human genes 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
- 108010015780 Viral Core Proteins Proteins 0.000 description 1
- 108010084938 adenovirus receptor Proteins 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 230000000240 adjuvant effect Effects 0.000 description 1
- 230000007815 allergy Effects 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
- 230000004075 alteration Effects 0.000 description 1
- 229940037003 alum Drugs 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 229940024545 aluminum hydroxide Drugs 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 239000003430 antimalarial agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 235000009697 arginine Nutrition 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 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 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229920006317 cationic polymer Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 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 1
- 230000009977 dual effect Effects 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002121 endocytic effect Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 231100000321 erythema Toxicity 0.000 description 1
- 210000003617 erythrocyte membrane Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- NBVXSUQYWXRMNV-UHFFFAOYSA-N fluoromethane Chemical compound FC NBVXSUQYWXRMNV-UHFFFAOYSA-N 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 229940093915 gynecological organic acid Drugs 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 102000027596 immune receptors Human genes 0.000 description 1
- 108091008915 immune receptors Proteins 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 238000002743 insertional mutagenesis Methods 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 244000000056 intracellular parasite Species 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000021633 leukocyte mediated immunity Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000001325 log-rank test Methods 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 210000004688 microtubule Anatomy 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- BSOQXXWZTUDTEL-ZUYCGGNHSA-N muramyl dipeptide Chemical compound OC(=O)CC[C@H](C(N)=O)NC(=O)[C@H](C)NC(=O)[C@@H](C)O[C@H]1[C@H](O)[C@@H](CO)O[C@@H](O)[C@@H]1NC(C)=O BSOQXXWZTUDTEL-ZUYCGGNHSA-N 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000007923 nasal drop Substances 0.000 description 1
- 229940100662 nasal drops Drugs 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 210000001623 nucleosome Anatomy 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 150000002895 organic esters Chemical class 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 238000012858 packaging process Methods 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 230000004526 pharmaceutical effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000008196 pharmacological composition Substances 0.000 description 1
- 229960000502 poloxamer Drugs 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229920000724 poly(L-arginine) polymer Polymers 0.000 description 1
- 108010011110 polyarginine Proteins 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229950008882 polysorbate Drugs 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 229940124551 recombinant vaccine Drugs 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 229940031439 squalene Drugs 0.000 description 1
- 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 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- LCJVIYPJPCBWKS-NXPQJCNCSA-N thymosin Chemical compound SC[C@@H](N)C(=O)N[C@H](CO)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CO)C(=O)N[C@H](CO)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@H]([C@H](C)O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@H](CCC(O)=O)C(O)=O LCJVIYPJPCBWKS-NXPQJCNCSA-N 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000005829 trimerization reaction Methods 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000003708 urethra Anatomy 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 239000000277 virosome Substances 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
- A61K39/002—Protozoa antigens
- A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
-
- 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
- A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10041—Use of virus, viral particle or viral elements as a vector
- C12N2710/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention relates to the field of medicine and biotechnology. More particularly, the invention relates to the use of capsid-modified adenoviral vectors to induce a potent immune response to a malaria parasite antigen such as Plasmodium circumsporozoite protein, which are suitable for vaccines against malaria.
- a malaria parasite antigen such as Plasmodium circumsporozoite protein
- Malaria is a severe disease that ranks among the most prevalent infections in tropical areas throughout the world. Approximately 300-500 million people become infected yearly, with relatively high rates of morbidity and mortality. Severe morbidity and mortality occur particularly in young children and in adults migrating to a malaria endemic area without having undergone prior malaria exposure.
- the World Health Organization (WHO) estimates that 2 - 3 million children die of malaria in Africa alone, every year. The widespread occurrence and the increasing incidence of malaria in many countries, caused by drug-resistant parasites (Plasmodium falciparum, recently also Plasmodium vivax) and insecticide-resistant vectors (Anopheles mosquitoes), underscore the need for developing new methods for the control of this disease
- Malaria parasites have a complicated life cycle consisting of pre- erythrocytic, erythrocytic and sexual parasitic forms, representing a potential target for the development of a malaria vaccine.
- the pre-erythrocytic and erythrocytic forms are found in the host, while the sexual forms occur in the vector.
- Immunization with live- attenuated irradiated sporozoites (IrSp) has been shown to induce sterile protection (i.e., complete resistance against parasite challenge) in mice (Nussenzweig et al. 1967), non-human primates (Gwadz et al. 1979) and human (Clyde et al. 1973, Edelman et al. 1993).
- IrSp IrSp Protection conferred by IrSp is mediated by sporozoite neutralization by both humoral (B cell) and cellular (T cell) immune responses (Tsuji et al. 2001 ).
- B cell humoral
- T cell cellular immune responses
- IrSp vaccination is an attractive solution, the only way to obtain sporozoites is by dissecting mosquito salivary glands, and there is currently no known technology to grow large numbers of sporozoites in vitro. Therefore, an alternate vaccine vector that can elicit an equally strong protective immunity against malaria is needed.
- CS circumsporozoite
- CS circumsporozoite
- NANP nuclear factor receptor
- T cell epitopes At the C-terminal region of the CS protein, there are several T cell epitopes, which can induce a significant cellular immune response (Tsuji et al. 2001 ).
- the humoral (antibody) response can eliminate parasites by interacting and neutralizing the infectivity of sporozoites (extra-cellular parasite) prior to entering hepatocyte, whereas the cellular (T cell) response can attack EEF (an intra-cellular parasite) by secreting interferon-gamma.
- EEF an intra-cellular parasite
- CS-based malarial vaccine that is currently undergoing human trials is GlaxoSmithKline's RTS, S, fusion protein of the Hepatitis B surface antigen and a portion of Plasmodium falciparum circumsporozoite protein (PfCSP) in a form of viruslike particle (International Patent Application No. PCT/EP1992/002591 to SmithKline Beecham Biologicals S.A., filed November 1 1 , 1992), has been shown to decrease malaria infection in clinical trials (Alonso et al. 2004, Alonso et al. 2005, Bejon et al. 2008).
- PfCSP Plasmodium falciparum circumsporozoite protein
- RTS induces an anti-PfCSP humoral immune response, but a relatively weak PfCSP-specific cellular (CD8+) response (Kester et al. 2008), which might be the reason for the relatively weak protection by RTS, S.
- adenovirus-based malaria vaccines can induce a protective cellular immune response (International Patent Application No. PCT/EP2003/051019, filed December 16, 2003, Rodrigues et al. 1997).
- B cell antigenic epitope e.g., a bacterial or viral epitope
- adenovirus capsid proteins such as Hexon, Fiber, Penton and pIX (Worgall et al. 2005, McConnell et al. 2006, Krause et al. 2006, Worgall et al. 2007, Shiratsuchi et al. 2010, Palma et al. 201 1 ).
- Ad5 Hexon which is the target capsid protein of neutralizing antibody
- that of other serotypes has also been constructed in order to escape pre-existing anti-Ad5 immunity (Wu et al. 2002, Roberts et al. 2006).
- the present disclosure relates to various adenovirus protein modifications to augment immune response to a transgene of a recombinant adenoviral vaccine and to circumvent pre-existing anti-adenovirus immunity.
- one embodiment is directed to a recombinant
- adenovirus derived from a recombinant adenovirus plasmid vector wherein the recombinant adenovirus plasmid vector comprises a nucleotide sequence encoding (i) a Plasmodium circumsporozoite protein, or antigenic portion thereof, operably linked to a heterologous promoter such as CMV or CMV5 and (ii) a modified capsid or core protein, wherein an immunogenic epitope of Plasmodium circumsporozoite has been inserted into or replaces at least part of a capsid or core protein.
- a heterologous promoter such as CMV or CMV5
- a modified capsid or core protein wherein an immunogenic epitope of Plasmodium circumsporozoite has been inserted into or replaces at least part of a capsid or core protein.
- the Plasmodium circumsporozoite protein further comprises a Plasmodium falciparum circumsporozoite protein.
- the circumsporozoite protein may further comprise a codon-optimized Plasmodium falciparum
- circumsporozoite protein and in some aspects, may be encoded by the sequence of SEQ ID NO:1 or SEQ ID NO:2.
- the immunogenic epitope further comprises a B cell epitope of Plasmodium circumsporozoite protein.
- the B cell epitope may be
- the capsid protein may comprise a Hexon hypervariable region (HVR).
- HVR may further comprise HVR1 or HVR5, wherein a portion of HVR1 or HVR5 is replaced with the B cell epitope.
- the capsid protein may further comprise a capsid Fiber protein wherein the B cell epitope is inserted into the Fiber protein.
- the B cell epitope is a Plasmodium falciparum circumsporozoite protein B cell epitope, wherein the B cell epitope is a repeat sequence, for example, (NANP) n (SEQ ID NO:17), wherein n is less than 40.
- the repeat sequence may be (NANP) 4 , (NANP) 6 , (NANP) 8 , (NANP)io, (NANP) 12 , (NANP) 14 , (NANP) 16 , (NANP) 18 , (NANP) 20 , (NANP) 22 , (NANP) 28 or (NANP) 34 .
- inventions are directed to a pharmaceutical composition or malaria vaccine composition comprising a recombinant adenovirus according to the above embodiments. Further embodiments include a method of treating, preventing, or diagnosing malaria, comprising administering a therapeutic amount of the
- a method for treatment comprising administering a prime-boost vaccination, wherein a subject is given a series of increasing dosages or same dosages at a given time interval.
- the time interval may be any length sufficient to generate a humoral and/or cellular immune response.
- the interval may be, but is not limited to, once every 3 weeks.
- Fig. 1 is a schematic diagram of a (NANP) n -HVR1 /PfCSP recombinant adenovirus in accordance with embodiments of the disclosure.
- Fig. 2 is a schematic diagram illustrating the preparation of the DNA fragment coding (NANP) 28 and HVR1 -flanking sequences by overlapping PCR in accordance with embodiments of the disclosure.
- Fig. 3 is a schematic diagram illustrating the preparation of the DNA fragment coding (NANP) 34 and HVR1 -flanking sequences by overlapping PCR in accordance with embodiments of the disclosure.
- Fig. 4 is a schematic diagram illustrating the preparation of the DNA fragment coding (NANP) 40 and HVR1 -flanking sequences by overlapping PCR in accordance with embodiments of the disclosure.
- Fig. 5 is a schematic diagram illustrating the construction of the (NANP) 4> io, i6, 22, or 28-HVR1 /PfCSP and (NANP) 2 2, or 28-HVR1 /CMV5-PfCSP in accordance with embodiments of the disclosure.
- Fig. 6 is a schematic diagram illustrating the construction of the (NANP) 34> or 40 -HVR1 /PfCSP and (NANP) 34 , 0 r 4o-HVR1 /CMV5-PfCSP. using pUC19-Mut plasmid in accordance with embodiments of the disclosure.
- Fig. 7 is the nucleic acid sequence of codon-optimized Plasmodium falciparum circumsporozoite protein (PfCSP, SEQ ID NO:1 ) and the corresponding amino acid sequence (SEQ ID NO:2).
- the inserted (NANP) 4 sequence is underlined.
- the inserted (NANP)-io sequence is underlined.
- the inserted (NANP) 6 sequence is underlined.
- the inserted (NANP) 2 2 sequence is underlined.
- the inserted (NANP) 2 s sequence is underlined.
- the inserted (NANP) 34 sequence is underlined.
- the inserted (NANP) 40 sequence is underlined.
- Fig. 15 shows the results of PCR analysis of the region where NANP repeats were inserted in (NANP) n -HVR1 /PfCSP. A 1 kb plus ladder (Invitrogen) was used as a molecular marker.
- Fig. 16 shows HVR1 -modified adenovirus growth after HVR1 -modified adenovirus DNA transfection of AD293 cells.
- the present inventors have found a novel recombinant adenovirus having a novel, capsid-modified structure that is derived from a recombinant adenovirus plasmid vector.
- the recombinant adenovirus is capable of infecting mammalian cells, causing the cells to express a Plasmodium circumsporozoite protein.
- the recombinant adenovirus also has a modified Hexon hypervariable region (HRV) sequence
- the recombinant adenovirus is obtained by the method of transfecting cells with the linearized recombinant adenovirus plasmid vector.
- the present inventors carried out extensive research on pharmaceuticals containing as an active ingredient a recombinant adenovirus having malaria infection preventive and therapeutic effects. As a result, the inventors found that the obtained recombinant adenovirus has the desired pharmaceutical effects. DETAILED DESCRIPTION
- a "nucleotide sequence,” “polynucleotide” or “DNA molecule” as contemplated by the current disclosure, may include double strand DNA or single strand DNA (i.e., a sense chain and an antisense chain constituting the double strand DNA), and is not limited to a full length thereof. Nucleotide sequences encoding an
- immunogenic foreign gene such as those disclosed herein below, encompass double strand DNA containing genomic DNA, single strand DNA (sense chain) containing cDNA, single strand DNA (antisense chain) having a sequence complementary to the sense chain, synthetic DNA, and fragments thereof, unless otherwise mentioned.
- Nucleotide sequences, polynucleotides or DNA molecules as used herein are not limited to the functional region, and may include at least one of an expression suppression region, a coding region, a leader sequence, an exon, and an intron.
- nucleotide sequences or polynucleotides may include RNA or DNA.
- a polypeptide containing a specific amino acid sequence and a polynucleotide containing a specific DNA sequence may include fragments, homologs, derivatives, and mutants of the polynucleotide. Examples of mutants of a nucleotide sequence or polynucleotide (such as mutant DNA), include naturally occurring allelic mutants;
- mutants having deletion, substitution, addition, and/or insertion. It should be understood that such mutants encode polypeptides having substantially the same function as the polypeptide encoded by the original non-mutated polynucleotide.
- the present disclosure relates to a recombinant adenovirus that can express an antigenic determinant of a Plasmodium parasite, and comprises one or more modified capsid and/or core proteins.
- the recombinant adenovirus is derived from a recombinant adenovirus plasmid vector, the generation of which is described in the Examples below. The use of adenovirus as a vector is discussed further below.
- the recombinant adenovirus plasmid vectors described herein may be used as a malaria vaccine or pharmaceutical composition, wherein both humoral and/or cellular immune responses against the Plasmodium parasite are induced.
- the Plasmodium parasite may be selected from any of the known
- Plasmodium (P.) species for example, P. falciparum, P. malariae, P. ovale, P. vivax, P. knowlesi, P. berghei, P. chabaudi and P. yoelii.
- the antigenic determinant is derived from the rodent-specific Plasmodium yoelii or the human-specific Plasmodium falciparum
- a recombinant adenovirus capsid-modified plasmid vector (also described as a recombinant adenovirus plasmid vector herein) is a plasmid that encodes and produces a capsid and/or core-modified recombinant adenovirus (also described as a recombinant adenovirus herein) that has a structure comprising one or more modified capsid and/or core proteins.
- the modification of the capsid and/or core proteins may be accomplished by insertion of at least one immunogenic epitope of a Plasmodium circumsporozoite protein.
- the capsid and/or core protein may be deleted and replaced by at least one immunogenic epitope of a Plasmodium circumsporozoite protein.
- the immunogenic epitope is a B-cell and/or T-cell epitope of a Plasmodium circumsporozoite protein.
- the addition of a B cell or T cell epitope may serve to enhance the efficacy of an adenoviral vector used as a malaria vaccine by establishing or enhancing the humoral immune response to the CS protein.
- the modified capsid and core proteins and their significance with respect to their use in the recombinant adenovirus described herein are discussed further below.
- the one or more modified capsid and/or core proteins may be a modified Hexon protein, a modified Fiber protein, a modified pVII protein or a combination thereof.
- a portion of a Hexon hypervariable region (HVR) is replaced by at least one B-cell epitope of a Plasmodium circumsporozoite protein.
- one or more B-cell and/or T cell epitope of a Plasmodium circumsporozoite protein may be inserted in the Fiber protein or Hexon HVR.
- the modified HVR may be HVR1 , HVR2, HVR3, HVR4, HVR5, HVR6 or HVR7.
- the modified HVR may be HVR1 or HVR5.
- such an HVR-modified Hexon may have a nucleic acid sequence of SEQ ID NO:3 (Fig. 8), SEQ ID NO:5 (Fig. 9), SEQ ID NO:7 (Fig. 10), SEQ ID NO:9 (Fig. 1 1 ), SEQ ID NO:1 1 (Fig. 12), SEQ ID NO:13 (Fig. 13), or SEQ ID NO:15 (Fig. 14).
- nucleic acid sequences encode modified capsid proteins having amino acid sequences of SEQ ID NO:4 (Fig. 8), SEQ ID NO:6 (Fig. 9), SEQ ID NO:8 (Fig. 10), SEQ ID NO:10 (Fig. 1 1 ), SEQ ID NO:12 (Fig. 12), SEQ ID NO:14 (Fig. 13), or SEQ ID NO:16 (Fig. 14), respectively.
- the HVR-modified Hexon may be produced by any suitable method known in the art, for example, a polymerase chain reaction (PCR) method.
- the HVR-modified Hexon may be produced by one or more PCR reactions to amplify a region of the hexon gene that contains Age I and Nde I restriction sites using primers that have an (NANP) n sequence (SEQ ID NO:17) instead of an HVR1 sequence.
- the HVR-modified Hexon is produced by an "overlapping" (or "overlap extension”) PCR method.
- Overlapping PCR a method similar to gene splicing for recombination of DNA, involves the use of oligonucleotide primers used in a PCR reaction to generate DNA fragments that have overlapping ends. The overlapping fragments are then combined in a fusion reaction wherein the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand, resulting in the production of a longer target (Horton et al. 1989).
- a T-cell epitope of a Plasmodium circumsporozoite protein may be inserted into an adenovirus core pVII protein at any of the following sites: the C-terminus, before the first Nuclear Localization Signal (NLS) or between the two NLS.
- a T-cell epitope of a Plasmodium circumsporozoite protein may replace a portion of the pVII protein.
- the recombinant adenovirus may express a transgenic protein or recombinant transgenic protein.
- the transgenic protein or recombinant transgenic protein is a Plasmodium circumsporozoite protein or an antigenic determinant that is encoded by a recombinant adenovirus plasmid vector as described herein, and is expressed by a recombinant adenovirus produced by said recombinant adenovirus plasmid vector after infection of one or more host cells.
- the recombinant adenovirus plasmid vectors comprise a nucleotide sequence encoding a recombinant transgenic protein.
- the recombinant transgenic protein may comprise an antigenic
- the antigenic determinant comprises a P. falciparum circumsporozoite gene (CS) protein or an antigenic portion thereof.
- the P. falciparum CS protein has demonstrated prevention of malaria when used as the basis of active immunization in humans against mosquito- borne infection.
- the antigenic determinant may further comprise an immunogenic epitope, such as a B cell and/or T cell epitope.
- the CS protein is codon-optimized for enhanced expression in a subject. Codon-optimization is based on the required amino acid content, the general optimal codon usage in the subject of interest as well as any aspects that should be avoided to ensure proper expression.
- Such aspects may be splice donor or acceptor sites, stop codons, polyadenylation (pA) signals, GC- and AT- rich sequences, internal TATA boxes, or any other aspects known in the art.
- the DNA sequence of the codon-optimized CS transgene is shown in Fig. 7 (SEQ ID NO:1 , P. falciparum).
- the recombinant adenovirus plasmid vector may be a modified P. falciparum recombinant adenovirus plasmid vectors, such as an HVR1 - modified adenovirus vector (e.g., (NANP) 4> 0 , 16, 22, 28, 34 or 40 -HVR1 /PfCSP or (NANP) 4> 0 , 16, 22, 28, 34 or 40-H VR1 /CMV5-Pf CSP).
- HVR1 - modified adenovirus vector e.g., (NANP) 4> 0 , 16, 22, 28, 34 or 40 -HVR1 /PfCSP or (NANP) 4> 0 , 16, 22, 28, 34 or 40-H VR1 /CMV5-Pf CSP.
- a recombinant adenovirus may be produced by one of the following modified P. falciparum recombinant adenovirus plasmid vectors: (NANP) 4 -HVR1 /PfCSP, (NANP) 10 -HVR1 /PfCSP, (NANP) 16 -HVR1 /PfCSP, (NANP) 22 - HVR1 /PfCSP, (NANP) 28 -HVR1 /PfCSP, (NANP) 4 -HVR1 /CMV5-PfCSP, (NANP) 10 - HVR1 /CMV5-PfCSP, (NANP) 16 -HVR1 /CMV5-PfCSP, (NANP) 22 -HVR1 /CMV5-PfCSP or (NANP) 28 -HVR1 /CMV5-PfCSP (Fig.
- the recombinant adenovirus may be produced in accordance with the methods described herein for producing a recombinant adenovirus plasmid vector with the ability to express a recombinant transgenic protein (e.g., Plasmodium CS protein) in
- Purification of a recombinant adenovirus may be performed by using known virus purification methods. For example, purification of 0.5 to 1 .0 ml_ of a stock virus obtained by the method of producing a recombinant adenovirus protein by inoculating insect cells (1 x 10 7 cells/10 cm dish), such as AD293 cells. The culture supernatant is then collected several days after the infection, and a virus pellet obtained by centrifugation is suspended in a buffer, such as PBS (Phosphate Buffered Saline). The resulting suspension is subjected to a sucrose gradient of 10 to 60% and then centrifuged (25,000 rpm for 60 minutes at 4°C) to collect a virus band. The collected virus is further suspended in PBS, subsequently centrifuged under the same conditions as above, and the resulting purified recombinant virus pellet is stored at 4°C in a buffer, such as PBS.
- a buffer such as PBS.
- an active ingredient of the pharmaceutical composition may comprise a recombinant adenovirus, which may be obtained by the genetic engineering techniques described herein. More specifically, the active ingredient may be a recombinant adenovirus comprising modified capsid and/or core proteins, wherein a portion of a Hexon hypervariable region (HVR), a portion of Fiber protein, a portion of pVII protein or a combination thereof is replaced by at least one immunogenic epitope of Plasmodium circumsporozoite protein.
- HVR Hexon hypervariable region
- circumsporozoite protein may be inserted in the Fiber protein, Hexon HVR or pVII protein.
- the recombinant adenovirus plasmid vector further comprises a transgenic protein or recombinant transgenic protein that is expressed by the recombinant adenovirus after infecting one or more host cells.
- recombinant transgenic protein may be a Plasmodium circumsporozoite protein or a malaria antigen of a Plasmodium circumsporozoite protein, wherein the malaria antigen comprises at least one immunogenic epitope (e.g., a B cell or T cell epitope) of
- the active ingredient of the pharmaceutical composition is a recombinant adenovirus derived from a recombinant adenovirus plasmid vector, wherein the recombinant adenovirus plasmid vector is one of the following modified P.
- falciparum recombinant adenovirus plasmid vectors (NANP) 4 - HVFM /PfCSP, (NANP) 10 -HVR1 /PfCSP, (NANP) 16 -HVR1 /PfCSP, (NANP) 22 - HVFM /PfCSP, (NANP) 28 -HVR1 /PfCSP, (NANP) 4 -HVR1 /CMV5-PfCSP, (NANP) 10 - HVR1 /CMV5-PfCSP, (NANP) 16 -HVR1 /CMV5-PfCSP, (NANP) 22 -HVR1 /CMV5-PfCSP or (NANP) 28 -HVR1 /CMV5-PfCSP (Fig.
- recombinant adenovirus plasmid vectors are capable of producing recombinant adenoviruses when transfected into cells (e.g., AD293 cells) and wherein the
- recombinant transgenic protein may be expressed in mammalian cells, including human cells.
- a pharmaceutical composition having an active ingredient is a recombinant adenovirus as described herein enhances malaria infection- preventing effects against a malaria infectious antigen and reduces the infectivity titer, as described further in the Examples below.
- the recombinant adenovirus may be used for the treatment of malaria infections associated with infection of target cells and tissues.
- target cells affected by such malaria infection include blood cells, hepatic cells, renal cells, brain cells, lung cells, epithelial cells, and muscular cells.
- tissues comprising such cells include the lung, liver, kidney, brain, arteries and veins, the stomach, intestines, urethra, skin, and muscle.
- the pharmaceutical composition may enhance malaria infection- preventing effects against infectious antigens, for example, malaria antigens such as sporozoite surface antigens (Circumsporozoite Protein (CSP) and
- the pharmaceutical composition may enhance malaria infection-preventing effects against a Plasmodium parasite, selected from any known Plasmodium (P) species, for example, P. falciparum, P. malariae, P. ovale, P. vivax, P. knowlesi, P. berghei, P. chabaudi and P.
- P Plasmodium
- a reduction of the infectivity titer by the pharmaceutical composition may result in an increased survival, disease-free survival, or infection-free survival period and survival, disease-free survival, or infection-free survival rate when compared to subjects not administered the
- the pharmaceutical composition is useful as a preventive or therapeutic agent for malaria infections caused by pathogens such as Plasmodium.
- the pharmaceutical composition is useful as a preventive or therapeutic agent for complications resulting from a malaria infection caused by pathogens such as Plasmodium.
- the infection-preventing effect of the recombinant adenovirus of the present invention in a subject can be provided, for example, by administering the pharmaceutical composition containing the capsid-modified recombinant adenovirus of the present invention and additives for pharmaceutical administration to vertebrates, particularly mammals, including humans, by intramuscular (i.m.), subcutaneous (s.c), intracutaneous (i.e.), intradermal (i.d.), intraperitoneal (i.p.), nasal, or respiratory route, and then immunizing the vertebrates with the pharmaceutical composition containing the recombinant adenovirus described herein as an active ingredient several times.
- a target pathogen such as a selected
- Plasmodium species may be compared with the survival rate, disease-free survival, or infection-free survival of subjects not given the pharmaceutical composition.
- the pharmaceutical composition may additionally comprise a pharmaceutically effective amount of capsid and/or core-modified recombinant adenovirus as described herein and a pharmaceutically acceptable carrier, which is described further below.
- an active ingredient of the vaccine composition may comprise a recombinant adenovirus, derived from a recombinant adenovirus plasmid vector as described herein. More specifically, the active ingredient may be a recombinant adenovirus comprising modified capsid or core proteins, wherein a portion of a Hexon hypervariable region (HVR), a portion of Fiber protein, a portion of pVII protein or a combination thereof are replaced by at least one immunogenic epitope of Plasmodium circumsporozoite protein.
- HVR Hexon hypervariable region
- the active ingredient of the vaccine composition may be derived from a recombinant adenovirus plasmid vector illustrated in Figs. 5-6, for example, (NANP) 4 - HVR1 /PfCSP, (NANP) 10 -HVR1 /PfCSP, (NANP) 16 -HVR1 /PfCSP, (NANP) 22 -
- HVR1 /PfCSP HVR1 /PfCSP
- (NANP) 28 -HVR1 /PfCSP (NANP) 4 -HVR1 /CMV5-PfCSP
- (NANP) 10 - HVR1 /CMV5-PfCSP (NANP) 16 -HVR1 /CMV5-PfCSP
- the vaccine composition when administered to a subject, first comprises a recombinant adenovirus having one or more antigenic portions of a Plasmodium CS protein (i.e., a B cell epitope, T cell epitope or both) inserted into or replacing at least a part of a capsid or core protein.
- a Plasmodium CS protein i.e., a B cell epitope, T cell epitope or both
- the vaccine composition may then express a recombinant transgenic protein, wherein the recombinant transgenic protein is a Plasmodium CS protein comprising a B cell epitope, T cell epitope or both.
- the antigenic portions of the Plasmodium CS protein are found in the recombinant transgenic protein and the modified capsid or core proteins promote or enhance acquired humoral immunity, cellular immunity, or both as described in the Examples below.
- the recombinant adenovirus as described herein is useful as a vaccine to promote or enhance humoral immunity, cellular immunity, or both.
- the vaccine composition may enhance infection- preventing effects against infectious antigens, for example, malaria antigens such as sporozoite surface antigens (CSP and TRAP) of malaria parasites, merozoite surface membrane protein MSPI, malaria S antigen secreted from erythrocytes infected with malaria, PfEMPI protein present in the knobs of erythrocytes infected with malaria,
- malaria antigens such as sporozoite surface antigens (CSP and TRAP) of malaria parasites, merozoite surface membrane protein MSPI, malaria S antigen secreted from erythrocytes infected with malaria, PfEMPI protein present in the knobs of erythrocytes infected with malaria,
- the vaccine composition is also useful as a preventive or therapeutic agent for malaria infections caused by pathogens such as Plasmodium.
- the vaccine composition is also useful as a preventive or therapeutic agent for complications resulting from a malaria infection by pathogens such as Plasmodium.
- a vaccine composition as described herein may comprise a
- a recombinant adenovirus as described herein and further comprising a pharmaceutically acceptable carrier according to a standard method.
- acceptable carriers include physiologically acceptable solutions, such as sterile saline and sterile buffered saline.
- the vaccine or pharmaceutical composition may be used in combination with a pharmaceutically effective amount of an adjuvant to enhance the anti-malaria effects.
- an adjuvant Any immunologic adjuvant that may stimulate the immune system and increase the response to a vaccine, without having any specific antigenic effect itself may be used as the adjuvant.
- Many immunologic adjuvants mimic evolutionarily conserved molecules known as pathogen-associated molecular patterns (PAMPs) and are recognized by a set of immune receptors known as Toll-like
- TLRs Receptors
- adjuvants examples include Freund's complete adjuvant, Freund's
- double stranded RNA a TLR3 ligand
- LPS LPS analogs such as monophosphoryl lipid A (MPL) (a TLR4 ligand), flagellin (a TLR5 ligand), lipoproteins, lipopeptides, single stranded RNA, single stranded DNA, imidazoquinolin analogs (TLR7 and TLR8 ligands), CpG DNA (a TLR9 ligand), Ribi's adjuvant
- the amount of adjuvant used can be suitably selected according to the degree of symptoms, such as softening of the skin, pain, erythema, fever, headache, and muscular pain, which might be expressed as part of the immune response in humans or animals after the administration of this type of vaccine.
- the vaccine or pharmaceutical composition described herein may be used in combination with other known pharmaceutical products, such as immune response-promoting peptides and antibacterial agents
- the vaccine or pharmaceutical composition may further comprise other drugs and additives.
- drugs or additives that may be used in conjunction with a vaccine or pharmaceutical composition described herein include drugs that aid intracellular uptake of the recombinant adenovirus or recombinant transgenic protein of the present invention, liposome and other drugs and/or additives that facilitate transfection, (e.g., fluorocarbon emulsifiers, cochleates, tubules, golden particles, biodegradable microspheres, and cationic polymers).
- the amount of the active ingredient contained in the vaccine or pharmaceutical composition described herein may be selected from a wide range of concentrations, Virus Particle Unit (VPU), Plaque Forming Unit (PFU), weight to volume percent (w/v %) or other quantitative measure of active ingredient amount, as long as it is a therapeutically or pharmaceutically effective amount.
- the dosage of the vaccine or pharmaceutical composition may be appropriately selected from a wide range according to the desired therapeutic effect, the administration method (administration route), the therapeutic period, the patient's age, gender, and other conditions, etc.
- the dosage of the recombinant adenovirus may be administered in an amount approximately corresponding to 10 2 to 10 14 PFU, preferably 10 5 to 10 12 PFU, and more preferably 10 6 to 10 10 PFU per patient, calculated as the PFU of the recombinant virus.
- the dosage when a recombinant adenovirus is administered to a subject as an active ingredient of the vaccine or pharmaceutical composition, may be selected from a wide range in terms of the amount of expressible DNA introduced into the vaccine host or the amount of transcribed RNA. The dosage also depends on the strength of the transcription and translation promoters used in any transfer vectors used.
- the vaccine composition or pharmaceutical composition described herein may be administered by directly injecting a recombinant adenovirus suspension prepared by suspending the recombinant adenovirus in PBS (phosphate buffered saline) or saline into a local site (e.g., into the lung tissue, liver, muscle or brain), by nasal or respiratory inhalation, or by intravascular (i.v.) (e.g., intraarterial, intravenous, and portal venous), subcutaneous (s.c), intracutaneous (i.e.), intradermal (i.d.), or intraperitoneal (i.p.) administration.
- the vaccine or pharmaceutical composition of the present invention may be administered more than once.
- one or more additional vaccinations may be given as a booster.
- One or more booster administrations can enhance the desired effect.
- booster immunization with a pharmaceutical composition containing the recombinant adenovirus as described herein may be performed.
- use of various other adjuvants, drugs or additives with the vaccine of the invention may enhance the therapeutic effect achieved by the administration of the vaccine or pharmaceutical composition.
- the pharmaceutically acceptable carrier may contain a trace amount of additives, such as substances that enhance the isotonicity and chemical stability.
- additives should be non-toxic to a human or other mammalian subject in the dosage and concentration used, and examples thereof include buffers such as phosphoric acid, citric acid, succinic acid, acetic acid, and other organic acids, and salts thereof;
- antioxidants such as ascorbic acid; low molecular weight (e.g., less than about 10 residues) polypeptides (e.g., polyarginine and tripeptide) proteins (e.g., serum albumin, gelatin, and immunoglobulin); amino acids (e.g., glycine, glutamic acid, aspartic acid, and arginine) ; monosaccharides, disaccharides, and other carbohydrates (e.g., cellulose and derivatives thereof, glucose, mannose, and dextrin) , chelating agents (e.g., EDTA); sugar alcohols (e.g., mannitol and sorbitol); counterions (e.g., sodium); nonionic surfactants (e.g., polysorbate and poloxamer) ; and PEG.
- polypeptides e.g., polyarginine and tripeptide proteins
- amino acids e.g., glycine, glutamic acid, aspartic acid
- the vaccine or pharmaceutical composition containing a recombinant adenovirus described herein may be stored as an aqueous solution or a lyophilized product in a unit or multiple dose container such as a sealed ampoule or a vial.
- Another embodiment further provides a method of preventing malaria infection, or a method of treating malaria comprising administering an effective amount of the recombinant adenoviral vaccine, formulation, or pharmaceutical composition.
- the present invention further provides a method of immunostimulation comprising administering an effective amount of a recombinant adenoviral vaccine composition, formulation, pharmaceutical composition or a combination thereof to a subject.
- Subjects may include humans, animals (such as mammals, birds, reptiles, fish, and amphibians), or any other subjects that may become infected with a malaria parasite.
- Malaria parasites may include a Plasmodium parasite, selected from any of known Plasmodium (P) species, for example, P. falciparum, P. malariae, P. ovale, P. vivax, P. knowlesi, P. berghei, P. chabaudi and P. yoelii.
- a recombinant adenovirus as described herein may be formed alone or may be together with a pharmaceutically acceptable carrier into a vaccine composition, formulation, or pharmaceutical composition, and administered to the subject.
- the administration route may be, for example, any administration route mentioned above.
- the pharmaceutically acceptable carrier for use in the present invention can be suitably selected from carriers commonly used in this technical field, according to the form of the pharmaceutical composition to be produced. For example, when the pharmacological composition is formed into an aqueous solution, purified water (sterile water) or a physiological buffer solution can be used as the carrier. When the pharmaceutical composition is formed into other appropriate solutions, organic esters capable of being injected, such as glycol, glycerol and olive oil may be used as the carrier.
- the composition may contain stabilizers, excipients and other commonly used substances in this technical field, and particularly in the field of vaccine
- the amount of recombinant adenovirus used in a vaccine composition, formulation, or pharmaceutical composition may be suitably selected from a wide range of concentrations, VPU, PFU, weight to volume percent (w/v %) or other quantitative measure of active ingredient amount.
- a suitable range of recombinant adenovirus in the composition is preferably about 0.0002 to about 0.2 (w/v %), and more preferably 0.001 to 0.1 (w/v %).
- the method of administration of a recombinant adenovirus vaccine composition, formulation, or pharmaceutical composition may be suitably selected according to the dosage form, the patient's age, gender and other conditions such as the severity of the disease.
- a suitable dosage form is a form for parenteral
- the composition can be intravenously administered and mixed with a replacement fluid such as a glucose solution or an amino acid solution as appropriate, or can be administered intramuscularly (i.m.),
- intracutaneously i.e.
- subcutaneously s.c.
- intradermal ⁇ i.d.
- intraperitoneal ⁇ i.p.
- the daily dosage of a recombinant adenovirus vaccine composition, formulation, or pharmaceutical composition may vary depending on the subject's condition, body weight, age, gender, etc.
- the dosage of a recombinant adenovirus is administered in an amount of approximately 0.001 to 100 mg per kg of body weight per day.
- the vaccine, formulation, or composition of the invention may be administered in one or more administrations per day.
- the dosage of the recombinant adenovirus is administered in an amount approximately corresponding to 10 2 to 10 14 PFU, preferably 10 5 to 10 12 PFU, and more preferably 10 6 to 10 10 PFU per patient, calculated as the PFU of the recombinant adenovirus particle.
- the vaccine composition of the present invention should be administered according to Good Medical Practice, considering the clinical condition (for example, the condition to be prevented or treated) of each patient, the delivery site of the vaccine composition containing the recombinant adenovirus, the target tissue, the administration method, the dosage regimen, and other factors known to those skilled in the art. Therefore, the proper dosage of the vaccine composition herein is determined in consideration of the above.
- Yet another embodiment of the disclosure relates to a method of treating or preventing a malaria infection in a subject, the method comprising administering an immunologic or therapeutic amount of a malaria vaccine composition comprising a recombinant adenovirus.
- the recombinant adenovirus of the malaria vaccine may comprise an antigenic determinant of a Plasmodium parasite, and may further comprise one or more modified capsid or core proteins.
- An immunologic, pharmacologic or therapeutic amount may be any suitable amount wherein a potent immune response is generated against one or more antigenic portions of the (CS) protein (i.e., the transgene, B cell epitope, or CD4+ T cell epitope) such that malarial infection is prevented or reduced in severity.
- the method of treating or preventing a malaria infection described above may comprise a priming step using a first recombinant adenovirus vector followed by one or more boosting steps using one or more different recombinant adenovirus vectors.
- This method may be used in subjects that have not yet been exposed to a wild-type adenovirus, or in a subject that has been previously exposed to a wild-type adenovirus vector, wherein the priming step recombinant adenovirus vector is used to circumvent existing adenovirus immunity. Further embodiments and examples are described below.
- Adenoviruses are non-enveloped DNA viruses comprising a set of viral capsid proteins (described below) and a viral genome, that have been widely used to deliver one or more therapeutic or antigenic transgene to a variety of cells in vitro and in vivo.
- serotype 5 (Ad5) is preferably used as a vector for foreign gene transduction because of its strong infectivity in vivo (Abbink et al. 2007). Expression of the antigenic transgene may be controlled by any promoter or enhancer element known in the art.
- Promoters which may be used to control gene expression include, but are not limited to, cytomegalovirus immediate early promoter (CMV), simian virus 40 (SV40) early promoter, cellular polypeptide chain elongation factor 1 alpha (EF1 ) promoter, Rous sarcoma virus (RSV) promoter, and tetracycline- regulated (TR) promoter.
- CMV cytomegalovirus immediate early promoter
- SV40 simian virus 40
- EF1 cellular polypeptide chain elongation factor 1 alpha
- RSV Rous sarcoma virus
- TR tetracycline- regulated
- a polyadenylation (pA) signal after the coding sequence may also be used for efficient transcription and translation.
- the recombinant adenovirus vector described herein may be replication-defective, having a deletion at least in the E1 region of the adenoviral genome, since the E1 region is required for replication, transcription, translation and
- the adenovirus (Ad) system is an attractive vector for the development of recombinant vaccines for a number of reasons.
- recombinant adenoviral vectors infect most mammalian cell types (both replicative and non- replicative), including, but not limited to, mouse and human cell types.
- the same vector may be used successfully in mouse models and human clinical trials alike.
- adenovirus has a high virion stability, 2) is well tolerated, 3) may be grown at high titer, 4) can accommodate large transgenes, 5) has a genome that has been extensively studied for many years such that the complete DNA sequence of several serotypes is known, facilitating the manipulation of the Ad genome by recombinant DNA techniques (Graham and Prevec 1992).
- the adenovirus vaccine platform is used as a viral vector for development of a vaccine that targets a pre-erythrocytic malaria parasite, and provides protection from malaria infection.
- known recombinant viral vectors Rodrigues et al. 1997, Bruna-Romero et al. 2001 , Anderson et al. 2004, Tao et al.
- adenovirus has been shown to be a suitable viral vector for a malaria vaccine because it can induce a strong protective cellular immune response to pre-erythrocytic malaria parasites (Rodrigues et al. 1997).
- the malaria parasite may be any one of the Plasmodium family.
- the targeted parasite may be P. yoelii or P. falciparum.
- Adenovirus vectors expressing PyCS as a transgene elicits a malaria- specific CD8+ T cell response
- Adenovirus is an attractive vector for inducing a significant CD8+ T cell- mediated protective immunity against malaria (Rodrigues et al. 1997, Rodrigues et al. 1998).
- the immunogenicity of a recombinant adenovirus expressing the P. yoelii (a rodent malaria parasite) CS protein, AdPyCS was determined using a rodent malaria model.
- the inoculation of mice with AdPyCS induces complete immunity in a significant proportion of mice, preventing the occurrence of parasitemia (Rodrigues et al. 1997).
- This protective effect is primarily mediated by CD8+ T cells, as evidenced by depletion of the T cell population and is corroborated by the fact that AdPyCS was unable to induce high titers of antibody response against malaria parasites.
- the shuttle vector may contain a GFP expression cassette and cloning sites for a transgene.
- the resulting shuttle vector (GFP/pShuttle-CMV) has dual pCMV promoters and SV40pAs for a transgene and GFP from pmaxGFP (Amaxa, Germany).
- the optimized PyCS fragment was inserted into Kpnl and Hindi 11 sites of GFP/pShuttle- CMV.
- Ad(PyCS+GFP) The immunogenicity of Ad(PyCS+GFP was determined by measuring the magnitude of the CS-specific CD8+ T cell response and the level of protective immunity against the plasmodial liver stages.
- Ad(PyCS+GFP) behaves equivalently to AdPyCS (Rodrigues et al 1997), and is a potentially useful tool in determining the in vivo tropism of AdPyCS.
- recombinant adenoviral vectors with modified capsid and core proteins were constructed to 1 ) enhance humoral immunity via B cell activation, 2) enhance humoral immunity via T helper cell activation, and 3) circumvent existing adenoviral immunity.
- Adenovirus is a non-enveloped naked double stranded DNA virus with an icosahedral shape, having 20 faces of equilateral triangles.
- the adenovirus capsid consists of 252 capsomers, of which 240 are Hexon trimers and 12 are penton pentamers.
- a secondary interaction occurs between the RGD (Asp-Arg-Gly) motif in the penton base with ⁇ 3, ⁇ 5 and similar integrins, facilitating subsequent internalization of adenovirus into the cell (Mathias et al. 1994, Wickham et al. 1993).
- adenovirus uses the coxsackie-adenovirus receptor, CAR, as a cellular receptor (Bergelson et al. 1997).
- CAR coxsackie-adenovirus receptor
- MHC class I molecules, VCAM, and heparan sulfate are shown to mediate attachment and entry of Ad5 (Chu et al. 2001 , Hong et al. 1997).
- Ad5 rapidly escapes from endocytic compartments into the cytosol (Meier and Greber 2003, Leopold and Crystal 2007).
- the virion then translocates to the nucleus using microtubules.
- the Fiber protein is shed as the earliest capsid protein in the process (Nakano et al.
- Adenoviruses of different serotypes demonstrate different trafficking patterns (Miyazawa et al. 1999, Miyazawa et al. 2001 ). Changing or modifying the Fiber protein can impact trafficking, which may be
- APC antigen presenting cells
- the adenovirus Fiber is a trimer divided into Fiber tail, shaft and knob domains (Henry et al. 1994, Rux and Burnett 2004, Chroboczek et al. 1995).
- the three dimensional structure of the knob domain is known, and together with mutagenesis studies, these studies allow the areas involved in CAR interaction and trimerization to be visualized (Kirby et al. 1999, Xia et al. 1995).
- the Fiber shaft projects from the virion and the Fiber knob contains the Coxsackie and Adenovirus Receptor (CAR) interaction domain (Roelvink et al. 1999, Bewley et al. 1999).
- CAR Coxsackie and Adenovirus Receptor
- the CAR-binding site of the Fiber knob consists primarily of residues from the AB loop and CD loop and extends secondarily to the FG and HI loop and the B, E and F ⁇ sheets (Roelvink et al. 1999, Bewley et al. 1999).
- the HI loop has been the best studied insertion site on the Fiber knob (Worgall et al. 2004, Mizuguchi and Hayakawa 2004, Koizumi et al. 2003,
- Hexon is the most abundant protein of the adenovirus capsid with 720 copies per virion. In the mature virus, Hexon exists as homotrimeric capsomeres which make up the facets of the icosahedral virion (Rux and Burnett 2004).
- adenovirus serotypes 2 and 5 (Ad2 and Ad5) Hexons have been solved, revealing a complex molecular architecture (Athapilly et al. 1994, Roberts et al. 1986, Rux and Burnett 2000).
- the base of each monomeric subunit consists of two beta- barrel motifs that are present in the capsid proteins of many icosahedral viruses. Three long loops (DE1 , FG1 , and FG2) extend out from the base structure to form the tower region of each molecule (Rux and Burnett 2004). Sequences within these loop domains protrude to the surface of the capsid to form the exterior of the virion.
- HVRs hypervariable regions
- HVRs were identified throughout the Hexon molecule (Crawford-Miksza and Schnurr 1996, Roberts et al 2006). Because the HVRs are poorly conserved between serotypes and do not appear to be involved in maintaining the structural integrity of Hexon, small changes could be made to these domains without affecting the viability of the virus (Rux and Burnett 2000). For example a hexahistidine tag can be inserted into HVR2, HVR3, HVR5, HVR6, and HVR7 without compromising virus viability (Wu et al. 2005).
- Hexon HVRs are often used as targets to efficiently induce an antibody response against peptides located in Hexon HVRs (Worgall et al. 2005, Crompton et al. 1994). Due to its poor conservation in length between serotypes and its position on the outermost surface of the adenovirus capsid (Rux and Burnett 2000, Crawford-Miksza and Schnurr 1996), Hexon HVR5 was initially chosen as a site for epitope insertion. Further, the crystal structure of Hexon indicates that HVR5 is a flexible loop on the capsid surface, suggesting that HVR5 can accommodate relatively large peptides without compromising the structural integrity of the capsid (Roberts et al. 1986).
- CD4+ and CD8+ epitopes have recently been identified (Leen et al. 2008), and the CD4+ T cell response to adenovirus is focused against conserved residues within the Hexon protein in humans (Onion et al. 2007, Heemskerk et al. 2006).
- the adenovirus core is composed of the viral genome and four core proteins.
- the terminal protein (TP) is covalently linked to the 5' end of each linear viral DNA strand at two copies per virion.
- Noncovalently and nonspecifically bound to the viral DNA through arginine-rich portions are three other core proteins mu ( ⁇ ), V (pV) and VII (pVII).
- pVII is the major core protein contributing roughly 700-800 copies per virion, and serves as a histone-like center around which viral DNA is wrapped to form nucleosome structures.
- circumsporozoite (CS) adenoviral vectors that have an immunodominant CS protein B epitope in an adenovirus capsid protein
- the transgene may be under a promoter such as CMV to augment cell-mediated and humoral immune responses to CS protein.
- a central repeat region is the conserved structure of CS protein among Plasmodium species, and antibody against this repeat sequence has been shown to have sporozoite neutralizing activity.
- Examples of a repeat sequence in Plasmodium CS protein are (NANP) n repeat (P. falciparum; SEQ ID NO:17), ANGAGNQPG repeat (P. v/Vax; SEQ ID NO:18) and NAAG repeat (P. malariae; SEQ ID NO:19), which can be inserted into adenovirus capsid proteins.
- NANP nucleic acid sequence
- (NANP)n repeats (SEQ ID NO:17) of PfCSP that may be inserted into HVR1 of adenovirus serotype 5 Hexon is more than 4.
- the number of (NANP)n repeats (SEQ ID NO:17) of PfCSP that may be inserted into HVR1 of adenovirus serotype 5 Hexon is less than 40.
- adenoviruses with forty repeats e.g., (NANP) 40 -HVR1 /PfCSP) did not show any growth, thereby indicating that the maximum number or NANP repeats that can be successfully inserted is forty.
- NANP nucleophilicity parameter sequence
- 4 to 39, 4 to 39, 5 to 39, 6 to 39, 7 to 39, 8 to 39, 9 to 39, 10 to 39, 1 1 to 39, 12 to 39, 13 to 39, 14 to 39, 15 to 39, 16 to 39, 17 to 39, 18 to 39, 19 to 39, 20 to 39, 21 to 39, 22 to 39, 23 to 39, 24 to 39, 25 to 39, 26 to 39, 27 to 39, 28 to 39, 29 to 39, 30 to 39, 31 to 39, 32 to 39, 33 to 39, 34 to 39, 35 to 39, 36 to 39, 37 to 39 or 38 to 39 (NANP) n repeats (SEQ ID NO:17) of PfCSP may be inserted into HVR1 of adenovirus serotype 5 Hexon.
- 22 to 34, 23 to 34, 24 to 34, 25 to 34, 26 to 34, 27 to 34, 28 to 34, 29 to 34, 30 to 34, 31 to 34, 32 to 34, 33 to 34 (NANP) n repeats (SEQ ID NO:17) of PfCSP may be inserted into HVR1 of adenovirus serotype 5 Hexon.
- the (NANP) n repeat sequence may be additionally inserted in the in the HI loop of Fiber.
- immunodominant neutralizing B cell epitopes to CS were mapped to develop improved CS protein adenovirus vaccines.
- mice immunized with recombinant P. falciparum CS protein (PyCS) generated high titers against the B cell epitope repeat, (NANP) n repeats (SEQ ID NO: 1
- adenovirus plasmid vector such as those described above (e.g., (NANP) n -HVR1 /PfCSP) shows a protective effect and improved infection-free survival upon challenge with malaria sporozoites (see Example 3, below).
- the B cell epitope repeat peptide should be presented on the surface of adenovirus virions so that immune system can recognize the epitope efficiently.
- Such insertion sites could be HVRs of Hexon and Loop structures in Fiber, and different insertion sites can be combined. Modification of adenovirus capsid and core proteins to enhance T helper cell activation
- a CD4+ epitope specific to the transgene used in an adenoviral vector may be incorporated into adenovirus proteins.
- the CD4+ epitope may be incorporated into pVII, pV and Hexon to augment immunogenicity of the adenoviral-based vaccine.
- Professional antigen presenting cells (APC) such as dendritic cells (DC) and B cells can uptake particulated pathogens like virus particles via endocytosis and present CD4+ epitopes in the pathogen to CD4+ T cells which acts as helper cells for humoral and/or cellular immune responses.
- pVII and Hexon may easily be used as adenovirus target proteins to insert antigenic CD4+ peptides because of high copy number of pVII (700-800 copies) and Hexon (720 copies) in one virion.
- adenovirus Fiber and Hexon capsid proteins may be modified to insert a B cell or T helper cell epitope to overcome existing immunity to adenovirus and/or enhance the humoral response to an adenovirus vaccine.
- An estimated 80% of young adults in human population have circulating neutralizing antibodies to adenovirus (Douglas 2007), especially to serotype 5 (Ad5).
- Ad5 serotype 5
- studies utilizing adenovirus as a gene therapy vector it was found that the presence of neutralizing antibodies in animals limits the expression of transgenes delivered by adenovirus.
- CD8+ T cell responses also contributed to the limitation of recombinant gene expression (Yang et al.
- Hexon is a major target for anti-Ad capsid immune responses (Roy et al. 2005, Wohlfart 1988), and is likely responsible for the potent adjuvant effect of adenovirus, including the induction of CD4+ and CD8+ T cell responses. Therefore, one strategy that has been employed to circumvent pre-existing anti-adenovirus immunity is to replace all or part of the Hexon with a different protein, for example, rare serotypes such as adenovirus 1 1 , 24, 26 and 35. Because Hexon is a major target of anti-adenovirus neutralizing antibody (Youil et al. 2002, Sumida et al. 2005), the entire Hexon or HVRs of Hexon may be swapped with the rare serotypes (Wu et al. 2002, Roberts et al. 2006).
- an adenoviral Hexon may be modified by replacement of HVR1 or HVR5 with an antigenic peptide to circumvent pre-existing anti-adenovirus immunity or anti-adenovirus neutralizing antibody induced by previous vaccination with adenoviral vector.
- an antigenic peptide may be an immunogenic epitope of Plasmodium CS protein, and in certain aspects, the epitope may comprise a central repeat sequence, CD4+ epitope sequence or CD8+ epitope sequence.
- Ad vaccines impede boosting of the vaccine by preventing expression and presentation of the antigen encoded by the transgene (Yang 1995, hackett et al. 2000, Harvey et al. 1999, Mastrangeli et al. 1996).
- FIG. 1 The structures of (NANP) n -HVR1 /PfCSP and (NANP) n -HVR1 /cmv5-PfCSP are shown in Fig. 1 .
- the adenovirus genome DNA used in the experiments is an E1 and E3- deleted human adenovirus serotype 5 from Stratagene (pAdEasy-1 ) and an adenovirus shuttle vector, pShuttle-CMV (Stratagene) was used to construct recombinant PfCSP adenovirus genome DNA.
- the pShuttle-CMV was modified to replace the CMV promoter region with the CMV5 promoter from pQBI-AdCMV5 (QBIOgene).
- the SgrA I- Kpn I fragment of pShuttle-CMV was replaced with the fragment containing the CMV5 promoter sequence and the upstream sequence from CMV promoter in pShuttle-CMV to construct pShuttle- CMV5.
- PfCSP amino acid sequence of P. falciparum 3D7 strain was used as a template sequence for codon-optimization for protein expression in human with Integrated DNA Technologies' (Coralville, IA USA) optimization software.
- DNA fragments that encode whole PfCSP except for the GPI-anchored motif at the C- terminus were synthesized by Integrated DNA Technologies.
- the synthesized PfCSP cDNA was cloned into Kpn I and Hind III sites in adenovirus shuttle vectors, pShuttle-CMV and pShuttle -CMV5 to construct PfCSP/ pShuttle-CMV and PfCSP/ pShuttle-CMV5.
- adenovirus shuttle vectors pShuttle-CMV and pShuttle -CMV5
- These recombinant shuttle vectors were linearized by Pme I digestion and used for homologous recombination with pAdEasy-1 vector in E. co// ' BJ5183 cells (Stratagene) to construct PfCSP the recombinant adenoviruses wt/PfCSP and wt/CMV5-PfCSP.
- DNA fragments containing (NANP) 4 , 0 , i 6 , 0 r 22-HVR1 were prepared by performing a two-step PCR method, resulting in the amplification of the region of the hexon gene that contains the Age I and Nde I sites using primers which have the (NANP) n sequence instead of HVR1 sequence.
- This PCR product was digested with Age I and Nde I, and used to replace the native Age l-Nde I region of the Sfi I - Sfi I fragment in a Sfi l/pUC19 vector, which contains the Sfi I - Sfi I fragment (about 6.6 kbp) from pAdEasy-1 in the multi-cloning site of pUC19.
- the sequence of the amplified region in the constructs was confirmed by sequencing.
- NANP repeats-HVFM was amplified by PCR with NANPF3 and HVR1 R1 primers using (NANP) 2 2-HVR1 /PfCSP as a template. These fragments were purified after agarose gel electrophoresis and used as templates for overlapping PCR with HVR1 F1 and HVR1 R1 primers at an annealing temperature of 58 °C. The overlapping PCR product was purified after agarose gel electrophoresis.
- the second overlapping PCR was performed using CD4HexF1 and CD4HexR1 primers to connect the DNA fragment containing the Age I site, the fragment coding (NANP) 28 repeats and the fragment containing the Nde I site.
- the resulting PCR product, the (NANP) 28 -HVR1 DNA fragment was purified and digested with Age I and Nde I restriction enzymes, and used to replace the Age l-Nde I region of hexon in Sfi l/pUC19 plasmid (Fig. 5). The sequence of the amplified region in the constructs was confirmed by sequencing. [00112]
- the DNA fragments containing (NANP) 34 or 4 o-HVR1 were prepared by overlapping PCR as shown in Figs.
- the first DNA fragments (Figs. 3 and 4; (I)), coding HVR1 -NANP repeats was amplified by PCR with HVR1 F1 and NANPR2 primers using (NANP) 22 -HVR1 /PfCSP as a template.
- the second DNA fragments (Figs. 3 and 4; (II)), coding NANP repeats was amplified by PCR with NANPF2 and NANPR4 primers using PfCSP/pShuttle-CMV as a template.
- NANP repeats-HVR1 was amplified by PCR with NANPF4 and HVR1 R1 primers using (NANP) 22 -HVR1 /PfCSP as a template. These fragments were purified after agarose gel electrophoresis and used as templates for overlapping PCR with HVR1 F1 and HVR1 R1 primers.
- an annealing temperature of 58 °C was used for the amplification of DNA fragments containing (NANP) 34 .
- an annealing temperature of 48 °C was used to amplify a wide range of (NANP) n repeats by miss- annealing.
- the overlapping PCR products were separated on agarose gel and gel corresponding to the estimated size was cut out for DNA purification.
- the second overlapping PCR was done as described above to connect the DNA fragment containing the Age I site (Figs. 3 and 4; (V)), the fragment coding (NANP) 34 or 4 o repeats (Figs. 3 and 4; (IV)) and the fragment containing the Nde I site (Figs. 3 and 4; (VI)).
- the PCR product was purified and digested with Age I and Nde I restrict enzymes, and used to replace the Age l-Nde I region of hexon in Sfi l/pUC19- Mut plasmid (Fig. 6).
- Sfi l/pUC19-Mut has a mutation at the Nde I site in pUC19 and used in order to increase ligation efficiency.
- the sequence of the amplified region in the constructs was confirmed by sequencing.
- NANP nucleophilicity parameter
- NANP nucleophilicity parameter function /PfCSP plasmids as templates.
- the region was amplified with HexF12 (GTGCTGGACATGGCTTCCACGTAC; SEQ ID NO:34) and Hex R1 3
- TTTAGGTGTTTGACCTTCGACACC SEQ ID NO:35 primers and the PCR products were analyzed on agarose gel. As shown in Fig. 15, the size of PCR products increased in association with the increase in NANP repeats, indicating that constructed adenovirus genome retains the inserted NANP repeats.
- Adenovirus genome DNA plasmid was linearized by Pac I digestion and used for transfection of AD293 cells (Stratagene) in order to produce recombinant adenovirus.
- Adenovirus seed solution was prepared from the transfected AD293 cells showing cytopathic effects (CPE) by several rounds of freeze/thaw and used for further virus amplification. Adenovirus growth was not observed in AD293 cells transfected with (NANP) 40 -HVR1 /PfCSP and (NANP) 40 -HVR1 /cmv5-PfCSP, suggesting that insertion of (NANP) 40 repeats in HVR1 of hexon deteriorated adenovirus fitness.
- CPE cytopathic effects
- adenovirus particles were purified by CsCI gradient centrifugation. The band was then collected and dialyzed against dialysis buffer (10 mM Tris-HCI, 150 mM NaCI, 10 mM MgCI 2 , 3% (w/v) Sucrose, pH7.8) to remove CsCI. Virus particle unit (v.p.) was calculated based on O.D. 2 eo
- Example 2 Evaluation of (NANP) n -HVR1/PfCSP adenovirus growth in vitro
- the quantity of adenovirus genomic DNA was measured after transfection of AD293 cells with the adenovirus genome DNA.
- AD293 cells were seeded into 24 well plates one day before transfection and linearized adenovirus genome DNA was transfected in triplicate using Lipofectamine 2000 (Invitrogen) at day 0.
- genomic DNA was extracted from transfected AD293 cells using QIAamp DNA Mini kit (QIAGEN).
- the copy number of adenovirus genomic DNA in the extracted DNA was measured by real time PCR using 7500 Real Time PCR system (Applied Biosystems).
- the primer set for the real time PCR reaction was 100KF (AACTTCTACCCCGTATTTGCC; SEQ ID NO:36) and 100KR
- the adenovirus growth showed a trend that was inversely correlated with number of NANP repeats inserted in HVR1 .
- a slight delay of adenovirus growth was observed in (NANP) 2 8-HVR1 /PfCSP-transfected wells and a significant delay in
- (NANP) 34 -HVR1 /PfCSP-transfected wells (Fig. 16). No increase in the (NANP) 40 - HVR1 /PfCSP-transfected wells was detected, nor was there any increase in the copy number of adenovirus genome DNA (Fig. 16), which is consistent with the fact we could not produce (NANP) 40 -HVR1 /PfCSP or (NANP) 40 -HVR1 /CMV5-PfCSP. These observations suggest that the maximum number of NANP repeats which can be inserted in HVR1 is less than forty.
- Example 3 Protection from blood stage malaria infection by
- the adjuvant (AS) was prepared by re-suspending Sigma
- Adjuvant System with 1 mL of PBS (-) containing 200 ⁇ g/mL saponin. Adenovirus and adenovirus solution was mixed with an equal amount of the adjuvant (AS) before the immunization.
- AS adjuvant
- a (NANP) 2 2- HVR1 /PfCSP adenovirus with the adjuvant (AS) induced much higher anti-NANP antibody titer than wt/PfCSP alone or wt/PfCSP with the adjuvant (AS) at week 9 (Fig. 17B).
- the resulting humoral response that was specific to the NANP repeat sequences was determined by ELISA. Briefly, five microliters of blood was collected from tail vein of the immunized mice and diluted in 495 ⁇ _ of PBS (-), and then the samples were centrifuged at 5,000 rpm for 5 min to prepare diluted plasma samples (x100).
- mice were intravenously challenged with 2,000 transgenic P. berghei sporozoites that express recombinant P. bergfre/ circumsporozoite protein having the PfCSP central repeat region (NANP repeats) instead of the original central repeat (Persson et al. 2002). Giemsa-stained blood smears were analyzed from 4 to 10 days after challenge to detect blood stage malaria parasite infection.
- mice in na ' ive, wt/PfCSP and wt/PfCSP with the adjuvant groups were infected by day 7, whereas 50% of mice immunized with (NANP) 2 2-HVR1 /PfCSP with the adjuvant were protected from blood stage malaria parasite infection (Fig. 17C).
- SAS software SAS Institute Japan, R9.1 ) was used for the statistical analysis.
- Example 4 Protection from blood stage malaria infection by
- NANP repeat-specific humoral response was determined by ELISA as described above. Using this immunization regimen, all (NANP) n -HVR1 /PfCSP adenoviruses induced a significantly higher anti-NANP antibody titer than wt/PfCSP at day 35 (Fig. 1 8B). The anti-NANP antibody titer in the na ' ive mice was lower than the minimum dilution (200-times dilution) and therefore was depicted as "2" in Figure 1 8B.
- lymphoblastoid cells The EMBO journal 16, 2294-2306 (1997). Bruna-Romero, O., Gonzalez-Aseguinolaza, G., Hafalla, J.C., Tsuji, M., and
- Single immunizing dose of recombinant adenovirus efficiently induces CD8+ T cell-mediated protective immunity against malaria.
- Neutralizing antibodies to adenovirus serotype 5 vaccine vectors are directed primarily against the adenovirus Hexon protein. J Immunol. 174:7179-71 85.
- CD4+ cytolytic T cell clone confers protection against murine malaria. J Exp Med. 172:1353-1 357.
Landscapes
- Health & Medical Sciences (AREA)
- Tropical Medicine & Parasitology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
La présente divulgation concerne des modifications de protéines d'adénovirus en vue d'augmenter la réponse immunitaire vis-à-vis d'un transgène d'un adénovirus recombinant et en vue de contourner l'immunité anti-adénovirus préexistante. Quelques modes de réalisation concernent un adénovirus recombinant dérivé d'un vecteur plasmidique d'adénovirus recombinant, comprenant une séquence nucléotidique codant pour un gène de protéine circumsporozoïte de Plasmodium ou pour une portion antigénique de celle-ci, liée de manière fonctionnelle à une séquence de promoteur hétérologue ; et une séquence modifiée de la région hypervariable de l'hexon (HRV) comprenant une séquence d'épitope de lymphocyte B immunogène d'une protéine circumsporozoïte de Plasmodium falciparum qui est insérée dans ou qui remplace au moins une partie de la séquence de HRV. D'autres modes de réalisation sont consacrés à une composition pharmaceutique ou à une composition de vaccin contre la malaria comprenant un adénovirus recombinant selon les modes de réalisation ci-dessus. Des modes de réalisation supplémentaires comprennent un procédé de traitement, de prévention ou de diagnostic de la malaria, comprenant l'administration d'une quantité thérapeutique de la composition pharmaceutique ou de la composition de vaccin contre la malaria conformément au mode de réalisation ci-dessus.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US2010/045952 WO2011022522A1 (fr) | 2009-08-18 | 2010-08-18 | Modification d'adénovirus recombinant par épitopes protéiques immunogènes de plasmodium circumsporozoïte |
USPCT/US2010/045952 | 2010-08-18 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2012023995A1 true WO2012023995A1 (fr) | 2012-02-23 |
Family
ID=45607431
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2011/025535 WO2012023995A1 (fr) | 2010-08-18 | 2011-02-18 | Modification d'une protéine de capside d'adénovirus recombinant par des épitopes immunogènes de protéine circumsporozoïte de plasmidium |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2012023995A1 (fr) |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105431535A (zh) * | 2013-06-03 | 2016-03-23 | Vlp治疗有限责任公司 | 疟疾疫苗 |
US9637532B2 (en) | 2013-07-12 | 2017-05-02 | Vlp Therapeutics, Llc | Virus like particle comprising PD-1 antigen or PD-1 ligand antigen |
WO2017125463A1 (fr) * | 2016-01-21 | 2017-07-27 | Janssen Vaccines & Prevention B.V. | Vaccin antipaludique amélioré de type adénoviral codant pour et présentant un antigène du paludisme |
US9969986B2 (en) | 2014-08-08 | 2018-05-15 | Vlp Therapeutics, Llc | Virus like particle comprising modified envelope protein E3 |
US10098943B2 (en) | 2014-09-11 | 2018-10-16 | Vlp Therapeutics, Llc | Flavivirus virus like particle |
US10385101B2 (en) | 2014-08-08 | 2019-08-20 | Vlp Therapeutics, Llc | Virus like particle comprising modified envelope protein E3 |
CN111108192A (zh) * | 2017-07-05 | 2020-05-05 | Nouscom股份公司 | 非人类人猿腺病毒核酸序列和氨基酸序列、含有其的载体及其用途 |
US11345726B2 (en) | 2012-02-16 | 2022-05-31 | VLP Theranentics. Inc. | Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV) virus-like particles comprising heterologous antigens inserted into the envelope protein |
US12134777B2 (en) | 2020-04-30 | 2024-11-05 | Vlp Therapeutics, Inc. | Alphavirus replicon vector and immunotherapy by administering same |
US12227770B2 (en) | 2017-12-20 | 2025-02-18 | Vlp Therapeutics, Inc. | Alphavirus replicon particle |
US12280102B2 (en) | 2020-04-17 | 2025-04-22 | Vlp Therapeutics, Inc. | Alphavirus replicon encoding chimeric SARS-CoV-2 receptor binding domains |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2008140474A1 (fr) * | 2006-10-26 | 2008-11-20 | Johns Hopkins University | Vaccins contre l'adénovirus recombinant |
US20090148477A1 (en) * | 2005-08-31 | 2009-06-11 | Genvec, Inc. | Adenoviral vector-based malaria vaccines |
US20090324702A1 (en) * | 2006-02-09 | 2009-12-31 | Shigeto Yoshida | Novel viral vector |
-
2011
- 2011-02-18 WO PCT/US2011/025535 patent/WO2012023995A1/fr active Application Filing
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20090148477A1 (en) * | 2005-08-31 | 2009-06-11 | Genvec, Inc. | Adenoviral vector-based malaria vaccines |
US20090324702A1 (en) * | 2006-02-09 | 2009-12-31 | Shigeto Yoshida | Novel viral vector |
WO2008140474A1 (fr) * | 2006-10-26 | 2008-11-20 | Johns Hopkins University | Vaccins contre l'adénovirus recombinant |
Non-Patent Citations (3)
Title |
---|
DATABASE GENBANK [online] 14 June 2008 (2008-06-14), "Human adenovirus 5 gene for hexon, complete cds, strain: Adenoid 75.", retrieved from http://www.ncbi.nlm.nih.gov/nuccore/AB330086 Database accession no. AB330086 * |
PALMA ET AL.: "Adenovirus particles that display the Plasmodium falciparum circumsporozoite protein NANP repeat induce sporozoite-neutralizing antibodies in mice.", VACCINE, vol. 29, no. 8, 11 February 2011 (2011-02-11), pages 1683 - 9 * |
SHIRATSUCHI ET AL.: "Replacing adenoviral vector HVR1 with a malaria B cell epitope improves immunogenicity and circumvents preexisting immunity to adenovirus in mice.", J CLIN INVEST, vol. 120, no. 10, 1 October 2010 (2010-10-01), pages 3688 - 701, XP055092318, DOI: doi:10.1172/JCI39812 * |
Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11345726B2 (en) | 2012-02-16 | 2022-05-31 | VLP Theranentics. Inc. | Chikungunya virus (CHIKV) or Venezuelan equine encephalitis virus (VEEV) virus-like particles comprising heterologous antigens inserted into the envelope protein |
CN105431535A (zh) * | 2013-06-03 | 2016-03-23 | Vlp治疗有限责任公司 | 疟疾疫苗 |
EP3004348A4 (fr) * | 2013-06-03 | 2016-08-03 | Vlp Therapeutics Llc | Vaccin contre le paludisme |
US9512190B2 (en) | 2013-06-03 | 2016-12-06 | VLP Thereapeutics, LLC | Malaria vaccine |
IL242656B (en) * | 2013-06-03 | 2022-07-01 | Vlp Therapeutics Inc | Malaria vaccine |
US10464986B2 (en) | 2013-07-12 | 2019-11-05 | Vlp Therapeutics, Llc | Virus like particle comprising PD-1 antigen or PD-1 ligand antigen |
US9637532B2 (en) | 2013-07-12 | 2017-05-02 | Vlp Therapeutics, Llc | Virus like particle comprising PD-1 antigen or PD-1 ligand antigen |
US10385101B2 (en) | 2014-08-08 | 2019-08-20 | Vlp Therapeutics, Llc | Virus like particle comprising modified envelope protein E3 |
US9969986B2 (en) | 2014-08-08 | 2018-05-15 | Vlp Therapeutics, Llc | Virus like particle comprising modified envelope protein E3 |
US10098943B2 (en) | 2014-09-11 | 2018-10-16 | Vlp Therapeutics, Llc | Flavivirus virus like particle |
US10517938B2 (en) | 2016-01-21 | 2019-12-31 | Janssen Vaccines & Prevention B.V. | Adenovirus based malaria vaccine encoding and displaying a malaria antigen |
WO2017125463A1 (fr) * | 2016-01-21 | 2017-07-27 | Janssen Vaccines & Prevention B.V. | Vaccin antipaludique amélioré de type adénoviral codant pour et présentant un antigène du paludisme |
CN111108192A (zh) * | 2017-07-05 | 2020-05-05 | Nouscom股份公司 | 非人类人猿腺病毒核酸序列和氨基酸序列、含有其的载体及其用途 |
CN111108192B (zh) * | 2017-07-05 | 2023-12-15 | Nouscom股份公司 | 非人类人猿腺病毒核酸序列和氨基酸序列、含有其的载体及其用途 |
US12227770B2 (en) | 2017-12-20 | 2025-02-18 | Vlp Therapeutics, Inc. | Alphavirus replicon particle |
US12280102B2 (en) | 2020-04-17 | 2025-04-22 | Vlp Therapeutics, Inc. | Alphavirus replicon encoding chimeric SARS-CoV-2 receptor binding domains |
US12134777B2 (en) | 2020-04-30 | 2024-11-05 | Vlp Therapeutics, Inc. | Alphavirus replicon vector and immunotherapy by administering same |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
RU2585228C2 (ru) | Модификация рекомбинантного аденовируса иммуногенными эпитопами белка circumsporozoite плазмодия | |
WO2012023995A1 (fr) | Modification d'une protéine de capside d'adénovirus recombinant par des épitopes immunogènes de protéine circumsporozoïte de plasmidium | |
JP2013502221A5 (fr) | ||
JP2022101574A (ja) | 組換えアデノウイルスおよびその使用 | |
JP6110347B2 (ja) | 新規方法および組成物 | |
ES2871907T3 (es) | Portadores de vacuna de adenovirus de chimpancé | |
KR101253363B1 (ko) | 개선된 아데노바이러스 벡터 및 그것의 용도 | |
JP5222289B2 (ja) | マラリア用ワクチン | |
US20140348791A1 (en) | Modified adenoviral vectors and methods of treatment using same | |
US20090110695A1 (en) | Compositions Comprising a Recombinant Adenovirus and an Adjuvant | |
US9555089B2 (en) | Modification of recombinant adenovirus with immunogenic plasmodium circumsporozoite protein epitopes | |
AU2006284756A2 (en) | Adenoviral vector-based malaria vaccines | |
JP2017186369A (ja) | 迅速かつ持続的な免疫学的治療法 | |
Palma et al. | Adenovirus particles that display the Plasmodium falciparum circumsporozoite protein NANP repeat induce sporozoite-neutralizing antibodies in mice | |
Bruder et al. | Molecular vaccines for malaria | |
AU2010286187B2 (en) | Modification of recombinant adenovirus with immunogenic Plasmodium circumsporozoite protein epitopes | |
Van Winkel et al. | Capsid-incorporation strategy to display antigens for an alternative adenoviral vector vaccine approach | |
JP2022551107A (ja) | アデノウイルスベクターおよびその使用 | |
EA047117B1 (ru) | Аденовирусные векторы и их применения | |
ES2377964T3 (es) | Vacunas contra la malaria basadas en virus recombinante |
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: 11818490 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 11818490 Country of ref document: EP Kind code of ref document: A1 |