NZ620242B2 - Influenza virus mutants and uses therefor - Google Patents
Influenza virus mutants and uses therefor Download PDFInfo
- Publication number
- NZ620242B2 NZ620242B2 NZ620242A NZ62024212A NZ620242B2 NZ 620242 B2 NZ620242 B2 NZ 620242B2 NZ 620242 A NZ620242 A NZ 620242A NZ 62024212 A NZ62024212 A NZ 62024212A NZ 620242 B2 NZ620242 B2 NZ 620242B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- virus
- m2ko
- δtm
- influenza
- seq
- Prior art date
Links
- 241000712461 unidentified influenza virus Species 0.000 title claims abstract description 73
- 241000700605 Viruses Species 0.000 claims abstract description 470
- 230000001902 propagating Effects 0.000 claims abstract description 5
- 210000004027 cells Anatomy 0.000 claims description 249
- 229960005486 vaccines Drugs 0.000 claims description 180
- 239000000203 mixture Substances 0.000 claims description 82
- 101700045377 mvp1 Proteins 0.000 claims description 81
- 208000006572 Human Influenza Diseases 0.000 claims description 60
- 206010022000 Influenza Diseases 0.000 claims description 59
- 230000035772 mutation Effects 0.000 claims description 49
- 241000712431 Influenza A virus Species 0.000 claims description 42
- 230000028993 immune response Effects 0.000 claims description 28
- 241000124008 Mammalia Species 0.000 claims description 22
- 230000001717 pathogenic Effects 0.000 claims description 22
- 210000003501 Vero Cells Anatomy 0.000 claims description 20
- 239000002245 particle Substances 0.000 claims description 17
- 238000000338 in vitro Methods 0.000 claims description 15
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 13
- 230000001809 detectable Effects 0.000 claims description 8
- 230000000644 propagated Effects 0.000 claims description 7
- 241000699802 Cricetulus griseus Species 0.000 claims description 6
- 210000001672 Ovary Anatomy 0.000 claims description 6
- 230000029812 viral genome replication Effects 0.000 claims description 4
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 claims 1
- 241000282339 Mustela Species 0.000 description 175
- 230000003612 virological Effects 0.000 description 98
- 230000037396 body weight Effects 0.000 description 78
- 238000011081 inoculation Methods 0.000 description 77
- 241001465754 Metazoa Species 0.000 description 71
- 210000002966 Serum Anatomy 0.000 description 70
- 101710043164 Segment-4 Proteins 0.000 description 69
- 101700038759 VP1 Proteins 0.000 description 69
- 101700005460 hemA Proteins 0.000 description 69
- 239000000185 hemagglutinin Substances 0.000 description 63
- 102000004965 antibodies Human genes 0.000 description 58
- 108090001123 antibodies Proteins 0.000 description 58
- 201000009910 diseases by infectious agent Diseases 0.000 description 50
- 238000002255 vaccination Methods 0.000 description 50
- 230000036760 body temperature Effects 0.000 description 46
- 239000002953 phosphate buffered saline Substances 0.000 description 45
- 102000005348 Neuraminidase Human genes 0.000 description 44
- 108010006232 Neuraminidase Proteins 0.000 description 44
- 230000002354 daily Effects 0.000 description 43
- 241000282341 Mustela putorius furo Species 0.000 description 42
- 150000007523 nucleic acids Chemical group 0.000 description 39
- 230000000694 effects Effects 0.000 description 38
- 210000004072 Lung Anatomy 0.000 description 34
- 102000004169 proteins and genes Human genes 0.000 description 34
- 108090000623 proteins and genes Proteins 0.000 description 34
- 230000002238 attenuated Effects 0.000 description 33
- 235000018102 proteins Nutrition 0.000 description 33
- 230000004083 survival Effects 0.000 description 30
- 210000001944 Turbinates Anatomy 0.000 description 28
- 239000000427 antigen Substances 0.000 description 27
- 102000038129 antigens Human genes 0.000 description 27
- 108091007172 antigens Proteins 0.000 description 27
- 108090001095 Immunoglobulin G Proteins 0.000 description 26
- 102000004851 Immunoglobulin G Human genes 0.000 description 26
- 108020004707 nucleic acids Proteins 0.000 description 26
- 102000011931 Nucleoproteins Human genes 0.000 description 25
- 108010061100 Nucleoproteins Proteins 0.000 description 25
- 230000002163 immunogen Effects 0.000 description 23
- 238000002965 ELISA Methods 0.000 description 21
- 229940027941 Immunoglobulin G Drugs 0.000 description 21
- 239000000243 solution Substances 0.000 description 21
- 229920000160 (ribonucleotides)n+m Polymers 0.000 description 19
- 102000004310 Ion Channels Human genes 0.000 description 19
- 108090000862 Ion Channels Proteins 0.000 description 19
- 210000002845 Virion Anatomy 0.000 description 19
- 200000000003 influenza A Diseases 0.000 description 19
- 239000002609 media Substances 0.000 description 19
- 238000000034 method Methods 0.000 description 19
- 229920001850 Nucleic acid sequence Polymers 0.000 description 18
- 230000002458 infectious Effects 0.000 description 18
- 229920001184 polypeptide Polymers 0.000 description 18
- 210000003491 Skin Anatomy 0.000 description 17
- 201000010099 disease Diseases 0.000 description 17
- 210000002345 respiratory system Anatomy 0.000 description 17
- 230000004044 response Effects 0.000 description 17
- 239000000523 sample Substances 0.000 description 17
- 239000006228 supernatant Substances 0.000 description 17
- 206010041232 Sneezing Diseases 0.000 description 16
- 230000000120 cytopathologic Effects 0.000 description 16
- 238000004519 manufacturing process Methods 0.000 description 16
- 241000197306 H1N1 subtype Species 0.000 description 15
- 239000000047 product Substances 0.000 description 15
- 210000001519 tissues Anatomy 0.000 description 15
- 210000000056 organs Anatomy 0.000 description 14
- 230000001086 cytosolic Effects 0.000 description 13
- 210000004369 Blood Anatomy 0.000 description 12
- 239000000443 aerosol Substances 0.000 description 12
- 238000004166 bioassay Methods 0.000 description 12
- 239000008280 blood Substances 0.000 description 12
- 230000003053 immunization Effects 0.000 description 12
- 230000000977 initiatory Effects 0.000 description 12
- 239000005723 virus inoculator Substances 0.000 description 12
- 101700044583 PB1 Proteins 0.000 description 11
- 108020000999 Viral RNA Proteins 0.000 description 11
- 230000035931 haemagglutination Effects 0.000 description 11
- 230000002633 protecting Effects 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- 230000004580 weight loss Effects 0.000 description 11
- UCSJYZPVAKXKNQ-HZYVHMACSA-N 1-[(1S,2R,3R,4S,5R,6R)-3-carbamimidamido-6-{[(2R,3R,4R,5S)-3-{[(2S,3S,4S,5R,6S)-4,5-dihydroxy-6-(hydroxymethyl)-3-(methylamino)oxan-2-yl]oxy}-4-formyl-4-hydroxy-5-methyloxolan-2-yl]oxy}-2,4,5-trihydroxycyclohexyl]guanidine Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 10
- 241000282414 Homo sapiens Species 0.000 description 10
- 108020004999 Messenger RNA Proteins 0.000 description 10
- 108010067390 Viral Proteins Proteins 0.000 description 10
- 102000016350 Viral Proteins Human genes 0.000 description 10
- 230000005540 biological transmission Effects 0.000 description 10
- 230000002708 enhancing Effects 0.000 description 10
- 230000012010 growth Effects 0.000 description 10
- 238000002649 immunization Methods 0.000 description 10
- 238000007918 intramuscular administration Methods 0.000 description 10
- 229920002106 messenger RNA Polymers 0.000 description 10
- 239000002773 nucleotide Substances 0.000 description 10
- 125000003729 nucleotide group Chemical group 0.000 description 10
- 230000000241 respiratory Effects 0.000 description 10
- 101710003000 ORF1/ORF2 Proteins 0.000 description 9
- 101700043858 PB2 Proteins 0.000 description 9
- 208000001756 Virus Disease Diseases 0.000 description 9
- 230000001413 cellular Effects 0.000 description 9
- 239000003795 chemical substances by application Substances 0.000 description 9
- 239000003814 drug Substances 0.000 description 9
- 230000001665 lethal Effects 0.000 description 9
- 231100000518 lethal Toxicity 0.000 description 9
- 239000000463 material Substances 0.000 description 9
- 230000002516 postimmunization Effects 0.000 description 9
- 241000271566 Aves Species 0.000 description 8
- YQEZLKZALYSWHR-UHFFFAOYSA-N Calypsol Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 8
- 241000371980 Influenza B virus (B/Shanghai/361/2002) Species 0.000 description 8
- 102000004389 Ribonucleoproteins Human genes 0.000 description 8
- 108010081734 Ribonucleoproteins Proteins 0.000 description 8
- 102100007241 ST6GAL1 Human genes 0.000 description 8
- 101710039861 ST6GAL1 Proteins 0.000 description 8
- BPICBUSOMSTKRF-UHFFFAOYSA-N Xylazine Chemical compound CC1=CC=CC(C)=C1NC1=NCCCS1 BPICBUSOMSTKRF-UHFFFAOYSA-N 0.000 description 8
- 229960001600 Xylazine Drugs 0.000 description 8
- 229920003013 deoxyribonucleic acid Polymers 0.000 description 8
- WSFSSNUMVMOOMR-UHFFFAOYSA-N formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 8
- 238000009472 formulation Methods 0.000 description 8
- 229960003299 ketamine Drugs 0.000 description 8
- 238000002941 microtiter virus yield reduction assay Methods 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 206010003694 Atrophy Diseases 0.000 description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 7
- 210000003734 Kidney Anatomy 0.000 description 7
- 238000011053 TCID50 method Methods 0.000 description 7
- 210000003437 Trachea Anatomy 0.000 description 7
- 230000000875 corresponding Effects 0.000 description 7
- 239000003599 detergent Substances 0.000 description 7
- 239000000499 gel Substances 0.000 description 7
- 230000002401 inhibitory effect Effects 0.000 description 7
- 239000011159 matrix material Substances 0.000 description 7
- 108010045030 monoclonal antibodies Proteins 0.000 description 7
- 102000005614 monoclonal antibodies Human genes 0.000 description 7
- 230000002829 reduced Effects 0.000 description 7
- 230000017613 viral reproduction Effects 0.000 description 7
- 235000019786 weight gain Nutrition 0.000 description 7
- 229920002676 Complementary DNA Polymers 0.000 description 6
- 210000003743 Erythrocytes Anatomy 0.000 description 6
- RFHAOTPXVQNOHP-UHFFFAOYSA-N Fluconazole Chemical compound C1=NC=NN1CC(C=1C(=CC(F)=CC=1)F)(O)CN1C=NC=N1 RFHAOTPXVQNOHP-UHFFFAOYSA-N 0.000 description 6
- 241000282412 Homo Species 0.000 description 6
- 210000003324 RBC Anatomy 0.000 description 6
- 210000001533 Respiratory Mucosa Anatomy 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 229940079593 drugs Drugs 0.000 description 6
- 230000036039 immunity Effects 0.000 description 6
- 230000036961 partial Effects 0.000 description 6
- 238000007920 subcutaneous administration Methods 0.000 description 6
- -1 substitutions Chemical class 0.000 description 6
- 230000001225 therapeutic Effects 0.000 description 6
- 210000000605 viral structures Anatomy 0.000 description 6
- 229940098773 Bovine Serum Albumin Drugs 0.000 description 5
- 108091003117 Bovine Serum Albumin Proteins 0.000 description 5
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 5
- 210000000987 Immune System Anatomy 0.000 description 5
- 241000713297 Influenza C virus Species 0.000 description 5
- 229960005322 Streptomycin Drugs 0.000 description 5
- 206010047461 Viral infection Diseases 0.000 description 5
- 238000007792 addition Methods 0.000 description 5
- 150000001413 amino acids Chemical class 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 230000004579 body weight change Effects 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 230000034994 death Effects 0.000 description 5
- 229960002518 gentamicin Drugs 0.000 description 5
- 239000001963 growth media Substances 0.000 description 5
- 230000028996 humoral immune response Effects 0.000 description 5
- 200000000004 influenza B Diseases 0.000 description 5
- 200000000005 influenza C Diseases 0.000 description 5
- 210000003292 kidney cell Anatomy 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- 210000004779 membrane envelope Anatomy 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000006011 modification reaction Methods 0.000 description 5
- 244000052769 pathogens Species 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000001681 protective Effects 0.000 description 5
- 238000001711 protein immunostaining Methods 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 206010064097 Avian influenza Diseases 0.000 description 4
- 241000282465 Canis Species 0.000 description 4
- 206010011224 Cough Diseases 0.000 description 4
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 4
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 4
- 229940088598 Enzyme Drugs 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000003886 Glycoproteins Human genes 0.000 description 4
- 108090000288 Glycoproteins Proteins 0.000 description 4
- 241000713196 Influenza B virus Species 0.000 description 4
- 210000004379 Membranes Anatomy 0.000 description 4
- 229960001412 Pentobarbital Drugs 0.000 description 4
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N Pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 4
- 101700077381 RNPC3 Proteins 0.000 description 4
- 210000000952 Spleen Anatomy 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 230000000240 adjuvant Effects 0.000 description 4
- 238000000137 annealing Methods 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 230000000534 elicitor Effects 0.000 description 4
- 210000003527 eukaryotic cell Anatomy 0.000 description 4
- 230000001605 fetal Effects 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 238000007489 histopathology method Methods 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 238000001802 infusion Methods 0.000 description 4
- 239000002054 inoculum Substances 0.000 description 4
- 238000001990 intravenous administration Methods 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 230000016379 mucosal immune response Effects 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 230000002194 synthesizing Effects 0.000 description 4
- 238000004448 titration Methods 0.000 description 4
- 229960001322 trypsin Drugs 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- RGCKGOZRHPZPFP-UHFFFAOYSA-N Alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 3
- 210000004556 Brain Anatomy 0.000 description 3
- 206010061428 Decreased appetite Diseases 0.000 description 3
- 208000000059 Dyspnea Diseases 0.000 description 3
- 206010013975 Dyspnoeas Diseases 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 206010015548 Euthanasia Diseases 0.000 description 3
- 229960002743 Glutamine Drugs 0.000 description 3
- 108090000745 Immune Sera Proteins 0.000 description 3
- 208000002979 Influenza in Birds Diseases 0.000 description 3
- 229960003971 Influenza vaccines Drugs 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- 231100000111 LD50 Toxicity 0.000 description 3
- 210000004185 Liver Anatomy 0.000 description 3
- 210000004940 Nucleus Anatomy 0.000 description 3
- 210000000956 Olfactory Bulb Anatomy 0.000 description 3
- 210000000496 Pancreas Anatomy 0.000 description 3
- 206010039101 Rhinorrhoea Diseases 0.000 description 3
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K Trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 3
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 3
- 229940088594 Vitamin Drugs 0.000 description 3
- 230000000890 antigenic Effects 0.000 description 3
- 235000019994 cava Nutrition 0.000 description 3
- 230000034303 cell budding Effects 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 238000007405 data analysis Methods 0.000 description 3
- 235000005911 diet Nutrition 0.000 description 3
- 230000037213 diet Effects 0.000 description 3
- 239000012470 diluted sample Substances 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 230000036449 good health Effects 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 238000010211 hemagglutination inhibition (HI) assay Methods 0.000 description 3
- 230000003067 hemagglutinative Effects 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 239000007927 intramuscular injection Substances 0.000 description 3
- 210000002429 large intestine Anatomy 0.000 description 3
- 231100000636 lethal dose Toxicity 0.000 description 3
- 101710030587 ligN Proteins 0.000 description 3
- 101700077585 ligd Proteins 0.000 description 3
- 230000000670 limiting Effects 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 210000004962 mammalian cells Anatomy 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 230000003448 neutrophilic Effects 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 239000008194 pharmaceutical composition Substances 0.000 description 3
- 229920000117 poly(dioxanone) Polymers 0.000 description 3
- 230000003389 potentiating Effects 0.000 description 3
- 238000009021 pre-vaccination Methods 0.000 description 3
- 230000035812 respiration Effects 0.000 description 3
- 230000029058 respiratory gaseous exchange Effects 0.000 description 3
- 238000011012 sanitization Methods 0.000 description 3
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 3
- 210000000813 small intestine Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 239000008399 tap water Substances 0.000 description 3
- 235000020679 tap water Nutrition 0.000 description 3
- 239000011778 trisodium citrate Substances 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 150000003722 vitamin derivatives Chemical class 0.000 description 3
- 229930003231 vitamins Natural products 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- BRZYSWJRSDMWLG-DJWUNRQOSA-N (2R,3R,4R,5R)-2-[(1S,2S,3R,4S,6R)-4,6-diamino-3-[(2S,3R,4R,5S,6R)-3-amino-4,5-dihydroxy-6-[(1R)-1-hydroxyethyl]oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H]([C@@H](C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-DJWUNRQOSA-N 0.000 description 2
- DKNWSYNQZKUICI-UHFFFAOYSA-N Amantadine Chemical compound C1C(C2)CC3CC2CC1(N)C3 DKNWSYNQZKUICI-UHFFFAOYSA-N 0.000 description 2
- 229960003805 Amantadine Drugs 0.000 description 2
- 241000700198 Cavia Species 0.000 description 2
- 210000000170 Cell Membrane Anatomy 0.000 description 2
- 229920001405 Coding region Polymers 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 108020004394 Complementary RNA Proteins 0.000 description 2
- 210000000805 Cytoplasm Anatomy 0.000 description 2
- 229940089114 Drug Delivery Device Drugs 0.000 description 2
- 102000033147 ERVK-25 Human genes 0.000 description 2
- 210000001163 Endosomes Anatomy 0.000 description 2
- 101700054771 GCA Proteins 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- WZUVPPKBWHMQCE-VYIIXAMBSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@@]2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-VYIIXAMBSA-N 0.000 description 2
- 108010059743 Influenza A virus M2 protein Proteins 0.000 description 2
- 101700061402 MTRX Proteins 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- MQUQNUAYKLCRME-INIZCTEOSA-N N-tosyl-L-phenylalanyl chloromethyl ketone Chemical compound C1=CC(C)=CC=C1S(=O)(=O)N[C@H](C(=O)CCl)CC1=CC=CC=C1 MQUQNUAYKLCRME-INIZCTEOSA-N 0.000 description 2
- 210000000440 Neutrophils Anatomy 0.000 description 2
- 206010030113 Oedema Diseases 0.000 description 2
- 229940049954 Penicillin Drugs 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 229960000380 Propiolactone Drugs 0.000 description 2
- 102000017143 RNA Polymerase I Human genes 0.000 description 2
- 108010013845 RNA Polymerase I Proteins 0.000 description 2
- 101710017884 Segment-8 Proteins 0.000 description 2
- 241000282898 Sus scrofa Species 0.000 description 2
- 230000024932 T cell mediated immunity Effects 0.000 description 2
- 241000710771 Tick-borne encephalitis virus Species 0.000 description 2
- 229960004854 VIRAL VACCINES Drugs 0.000 description 2
- 231100000494 adverse effect Toxicity 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000000569 anti-influenza Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 229960000626 benzylpenicillin Drugs 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000036755 cellular response Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 239000003184 complementary RNA Substances 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 230000001419 dependent Effects 0.000 description 2
- 230000000881 depressing Effects 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 230000012202 endocytosis Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 239000003701 inert diluent Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 238000010255 intramuscular injection Methods 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000008297 liquid dosage form Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000001404 mediated Effects 0.000 description 2
- 229960004927 neomycin Drugs 0.000 description 2
- 230000001264 neutralization Effects 0.000 description 2
- 239000012457 nonaqueous media Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 239000000902 placebo Substances 0.000 description 2
- 229940068196 placebo Drugs 0.000 description 2
- 238000003825 pressing Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- DNIAPMSPPWPWGF-UHFFFAOYSA-N propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 230000001932 seasonal Effects 0.000 description 2
- 125000005629 sialic acid group Chemical group 0.000 description 2
- 108010061514 sialic acid receptor Proteins 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 230000001052 transient Effects 0.000 description 2
- 230000003442 weekly Effects 0.000 description 2
- 230000004584 weight gain Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- VEZXCJBBBCKRPI-UHFFFAOYSA-N β-Propiolactone Chemical compound O=C1CCO1 VEZXCJBBBCKRPI-UHFFFAOYSA-N 0.000 description 2
- SILNNFMWIMZVEQ-UHFFFAOYSA-N 1,3-dihydrobenzimidazol-2-one Chemical compound C1=CC=C2NC(O)=NC2=C1 SILNNFMWIMZVEQ-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-Tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 210000000683 Abdominal Cavity Anatomy 0.000 description 1
- 229940023040 Acyclovir Drugs 0.000 description 1
- 229960001280 Amantadine Hydrochloride Drugs 0.000 description 1
- 206010059512 Apoptosis Diseases 0.000 description 1
- 241000701465 BK virus strain GS Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108010004032 Bromelains Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 229960001631 Carbomer Drugs 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 108010008978 Chemokine CXCL10 Proteins 0.000 description 1
- 102000006579 Chemokine CXCL10 Human genes 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- GZCGUPFRVQAUEE-KCDKBNATSA-N D-(+)-Galactose Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-KCDKBNATSA-N 0.000 description 1
- 210000004443 Dendritic Cells Anatomy 0.000 description 1
- 229940009976 Deoxycholate Drugs 0.000 description 1
- 210000001198 Duodenum Anatomy 0.000 description 1
- 108010092799 EC 2.7.7.49 Proteins 0.000 description 1
- 101700079760 EFCB Proteins 0.000 description 1
- 101710005090 ERVFC1-1 Proteins 0.000 description 1
- 101710013371 ERVS71-1 Proteins 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- LVGKNOAMLMIIKO-QXMHVHEDSA-N Ethyl oleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OCC LVGKNOAMLMIIKO-QXMHVHEDSA-N 0.000 description 1
- ZJAOAACCNHFJAH-UHFFFAOYSA-N Foscarnet Chemical compound OC(=O)P(O)(O)=O ZJAOAACCNHFJAH-UHFFFAOYSA-N 0.000 description 1
- 229960002963 Ganciclovir Drugs 0.000 description 1
- IRSCQMHQWWYFCW-UHFFFAOYSA-N Ganciclovir Chemical compound O=C1NC(N)=NC2=C1N=CN2COC(CO)CO IRSCQMHQWWYFCW-UHFFFAOYSA-N 0.000 description 1
- 206010069767 H1N1 influenza Diseases 0.000 description 1
- 241000197304 H2N2 subtype Species 0.000 description 1
- 241000252870 H3N2 subtype Species 0.000 description 1
- 206010019233 Headache Diseases 0.000 description 1
- 208000002672 Hepatitis B Diseases 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010022005 Influenza viral infection Diseases 0.000 description 1
- 102000003996 Interferon beta Human genes 0.000 description 1
- 108090000467 Interferon beta Proteins 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 229960001388 Interferon-beta Drugs 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000008070 Interferon-gamma Human genes 0.000 description 1
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 1
- 241000229754 Iva xanthiifolia Species 0.000 description 1
- 210000004731 Jugular Veins Anatomy 0.000 description 1
- 210000001821 Langerhans Cells Anatomy 0.000 description 1
- 206010024264 Lethargy Diseases 0.000 description 1
- 108010028921 Lipopeptides Proteins 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 241000282560 Macaca mulatta Species 0.000 description 1
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 229960003152 Metisazone Drugs 0.000 description 1
- 210000004400 Mucous Membrane Anatomy 0.000 description 1
- 210000003928 Nasal Cavity Anatomy 0.000 description 1
- 208000008721 Needlestick Injury Diseases 0.000 description 1
- 241000772415 Neovison vison Species 0.000 description 1
- 108010061543 Neutralizing Antibodies Proteins 0.000 description 1
- 229960005030 OTHER VACCINES in ATC Drugs 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 241000061984 Orinoco virus Species 0.000 description 1
- 241000712464 Orthomyxoviridae Species 0.000 description 1
- 101710027500 PBRM1 Proteins 0.000 description 1
- 101700058227 POLI Proteins 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 108091005771 Peptidases Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 241001205902 Qualyub virus Species 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N RIFAMPICIN Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 238000010240 RT-PCR analysis Methods 0.000 description 1
- 206010037742 Rabies Diseases 0.000 description 1
- 229960000329 Ribavirin Drugs 0.000 description 1
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 1
- 229940081190 Rifampin Drugs 0.000 description 1
- UBCHPRBFMUDMNC-UHFFFAOYSA-N Rimantadine Chemical compound C1C(C2)CC3CC2CC1(C(N)C)C3 UBCHPRBFMUDMNC-UHFFFAOYSA-N 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 210000004927 Skin cells Anatomy 0.000 description 1
- 229920000978 Start codon Polymers 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 102400000368 Surface protein Human genes 0.000 description 1
- 210000001744 T-Lymphocytes Anatomy 0.000 description 1
- FPKOPBFLPLFWAD-UHFFFAOYSA-N Trinitrotoluene Chemical compound CC1=CC=C([N+]([O-])=O)C([N+]([O-])=O)=C1[N+]([O-])=O FPKOPBFLPLFWAD-UHFFFAOYSA-N 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 108010001801 Tumor Necrosis Factor-alpha Proteins 0.000 description 1
- DLGSOJOOYHWROO-WQLSENKSSA-N [(Z)-(1-methyl-2-oxoindol-3-ylidene)amino]thiourea Chemical compound C1=CC=C2N(C)C(=O)\C(=N/NC(N)=S)C2=C1 DLGSOJOOYHWROO-WQLSENKSSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical class [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 229960004150 aciclovir Drugs 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid Chemical compound OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- MKUXAQIIEYXACX-UHFFFAOYSA-N acyclovir Chemical compound N1C(N)=NC(=O)C2=C1N(COCCO)C=N2 MKUXAQIIEYXACX-UHFFFAOYSA-N 0.000 description 1
- 230000001058 adult Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 229930002945 all-trans-retinaldehyde Natural products 0.000 description 1
- 201000005794 allergic hypersensitivity disease Diseases 0.000 description 1
- WOLHOYHSEKDWQH-UHFFFAOYSA-N amantadine hydrochloride Chemical compound [Cl-].C1C(C2)CC3CC2CC1([NH3+])C3 WOLHOYHSEKDWQH-UHFFFAOYSA-N 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 229940121363 anti-inflammatory agents Drugs 0.000 description 1
- 230000002155 anti-virotic Effects 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 230000003115 biocidal Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229960000074 biopharmaceuticals Drugs 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 101700086494 buk1 Proteins 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000001364 causal effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 230000000973 chemotherapeutic Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000000295 complement Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002596 correlated Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000004748 cultured cells Anatomy 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-M deoxycholate Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-M 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 230000003292 diminished Effects 0.000 description 1
- 238000003618 dip coating Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002255 enzymatic Effects 0.000 description 1
- 210000003426 epidermal Langerhans cell Anatomy 0.000 description 1
- 229940093471 ethyl oleate Drugs 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 238000005755 formation reaction Methods 0.000 description 1
- 229960005102 foscarnet Drugs 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 108010074605 gamma-Globulins Proteins 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000002068 genetic Effects 0.000 description 1
- 150000004676 glycans Polymers 0.000 description 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-N guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 231100001045 histological change Toxicity 0.000 description 1
- 230000036732 histological change Effects 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000003308 immunostimulating Effects 0.000 description 1
- 230000001861 immunosuppresant Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 230000000415 inactivating Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 200000000012 influenza A (H3N2) Diseases 0.000 description 1
- 229940117432 influenza B virus antigen Drugs 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 238000010212 intracellular staining Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000002045 lasting Effects 0.000 description 1
- 229960001226 live attenuated influenza Drugs 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 201000005296 lung carcinoma Diseases 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 230000003278 mimic Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 102000035365 modified proteins Human genes 0.000 description 1
- 108091005569 modified proteins Proteins 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002417 nutraceutical Substances 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Polymers 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 150000002895 organic esters Chemical class 0.000 description 1
- 101700051975 pbs-5 Proteins 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- XUYJLQHKOGNDPB-UHFFFAOYSA-N phosphonoacetic acid Chemical compound OC(=O)CP(O)(O)=O XUYJLQHKOGNDPB-UHFFFAOYSA-N 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 101700016463 pls Proteins 0.000 description 1
- 229920001983 poloxamer Polymers 0.000 description 1
- 229920001888 polyacrylic acid Polymers 0.000 description 1
- 230000001402 polyadenylating Effects 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000023 polynucleotide Polymers 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002335 preservative Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000000770 pro-inflamatory Effects 0.000 description 1
- 230000000750 progressive Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000001737 promoting Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 230000002685 pulmonary Effects 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 230000010837 receptor-mediated endocytosis Effects 0.000 description 1
- 230000001105 regulatory Effects 0.000 description 1
- 230000003362 replicative Effects 0.000 description 1
- 230000002207 retinal Effects 0.000 description 1
- 235000020945 retinal Nutrition 0.000 description 1
- 239000011604 retinal Substances 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 231100000245 skin permeability Toxicity 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 201000010740 swine influenza Diseases 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 230000002588 toxic Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 210000003412 trans-Golgi Network Anatomy 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 238000001039 wet etching Methods 0.000 description 1
- 239000000080 wetting agent 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
-
- 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
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
-
- 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
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/543—Mucosal route intranasal
-
- 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
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
-
- 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- 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
- A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/70—Multivalent vaccine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/76—Viruses; Subviral particles; Bacteriophages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/145—Orthomyxoviridae, e.g. influenza virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16122—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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16151—Methods of production or purification of viral material
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16161—Methods of inactivation or attenuation
- C12N2760/16162—Methods of inactivation or attenuation by genetic engineering
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16171—Demonstrated in vivo effect
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16211—Influenzavirus B, i.e. influenza B virus
- C12N2760/16234—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16211—Influenzavirus B, i.e. influenza B virus
- C12N2760/16261—Methods of inactivation or attenuation
- C12N2760/16262—Methods of inactivation or attenuation by genetic engineering
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
Abstract
Disclosed is a recombinant influenza virus having a mutant M gene comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, and a method for propagating such viruses. The sequences are as defined in the specification.
Description
INFLUENZA VIRUS MUTANTS AND USES THEREFOR
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 61/501,034, filed June
24, 2011, the content of which is incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0000] The instant application contains a Sequence Listing which has been submitted in ASCII
format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy,
created on February 21, 2013, is named 090248-0125_SL.txt and is 28,140 bytes in size.
BACKGROUND
[0001] Influenza is a leading cause of death among American adults. Each year, about 36,000
people die from influenza, and more than 200,000 people are hospitalized. Influenza is a highly
contagious disease that is spread by coughing, sneezing and through direct physical contact with
objects that carry the virus such as doorknobs and telephones. Symptoms of influenza include
fever, extreme fatigue, headache, chills and body aches; about 50 percent of infected people have
no symptoms but are still contagious. Immunization is 70-90 percent effective in preventing
influenza in healthy people under the age of 65, as long as the antigenicities of the circulating
virus strain match those of the vaccine.
[0002] Vaccination is the main method for preventing influenza, and both live attenuated and
inactivated (killed) virus vaccines are currently available. Live virus vaccines, typically
administered intranasally, activate all phases of the immune system and can stimulate an immune
response to multiple viral antigens. Thus, the use of live viruses overcomes the problem of
destruction of viral antigens that may occur during preparation of inactivated viral vaccines. In
addition, the immunity produced by live virus vaccines is generally more durable, more
2
48204255.2
effective, and more cross-reactive than that induced by inactivated vaccines, and live virus
vaccines are less costly to produce than inactivated virus vaccines. However, the mutations in
attenuated virus are often ill-defined, and reversion is a concern.
SUMMARY
[0003] In one aspect, the invention provides a recombinant influenza virus having a mutant M
gene comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
[0004] In another aspect, the invention provides a composition comprising: a recombinant
influenza virus having a mutant M gene comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID
NO:3.
[0005] In another aspect, the invention provides a method for propagating a recombinant
influenza virus, comprising: contacting an isolated host cell with a recombinant influenza virus
comprising SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO: 3; and incubating the host cell for a
sufficient time and under conditions suitable for viral replication, wherein the host cell is
modified to produce a wild-type version of the influenza M gene, thereby providing the gene
product to the virus in trans.
[0006] In another aspect, the invention provides a recombinant influenza virus when
propagated by a method of the invention.
[0007] Certain statements that appear below are broader than what appears in the statements of
the invention above. These statements are provided in the interests of providing the reader with
a better understanding of the invention and its practice. The reader is directed to the
accompanying claim set which defines the scope of the invention.
[0008] Also described is a nucleic acid sequence comprising SEQ ID NO:1.
[0009] Also described is a nucleic acid sequence comprising SEQ ID NO:2.
3
48204255.2
[0010] Also described is a nucleic acid sequence comprising SEQ ID NO:3.
[0011] Also described is a composition comprising SEQ ID NO:1, SEQ ID NO:2 or SEQ ID
NO: 3, operably linked to (i) a promoter, and (ii) a transcription termination sequence.
[0012] Also described is a recombinant influenza virus comprising a mutation in the M gene.
In some embodiments, the recombinant influenza virus comprises SEQ ID NO:1, SEQ ID NO:2,
or SEQ ID NO: 3. In some embodiments, the mutation in the M gene results in failure of the
virus to express the M2 protein, or causes the virus to express a truncated M2 protein having the
amino acid sequence of SEQ ID NO:4. In some embodiments, the mutation in the M gene does
not revert to wild-type or to a non-wild-type sequence encoding a functional M2 protein for at
least 10 passages in an in vitro host cell system. In some embodiments, the virus is an influenza
A virus. In some embodiments, the virus is non-pathogenic in a mammal infected with the virus.
In some embodiments, the in vitro cell system comprises Chinese Hamster Ovary cells. In some
embodiments, the in vitro cell system comprises Vero cells.
[0013] Also described is a cell comprising the recombinant influenza virus of any one of
claims 5-10. In some embodiments, the cell is in vitro. In some embodiments, the cell is in vivo.
[0014] Also described is a composition comprising: a recombinant influenza virus comprising
a mutation in the M gene. In some embodiments, the composition comprises SEQ ID NO:1,
SEQ ID NO: 2, or SEQ ID NO:3. In some embodiments, the mutation in the M gene results in
failure of the virus to express the M2 protein, or causes the virus to express a truncated M2
protein having the amino acid sequence of SEQ ID NO:4. In some embodiments, the virus is an
influenza A virus. In some embodiments, the composition is non-pathogenic to a mammal
administered the composition. In some embodiments, the composition elicits a detectable
immune response in a mammal within about three weeks after administration of the composition
to the mammal.
4
48204255.2
[0015] Also described is a vaccine comprising: a recombinant influenza virus comprising a
mutation in the M gene. In some embodiments, the vaccine comprises SEQ ID NO:1, SEQ ID
NO:2, or SEQ ID NO: 3. In some embodiments, the mutation in the M gene results in failure of
the virus to express the M2 protein, or causes the virus to express a truncated M2 protein having
the amino acid sequence of SEQ ID NO:4. In some embodiments, the virus is an influenza A
virus. In some embodiments, the vaccine is non-pathogenic to a mammal administered the
vaccine. In some embodiments, the vaccine elicits a detectable immune response in a mammal
within about three weeks after administration of the vaccine to the mammal. In some
embodiments, the vaccine comprises at least two different influenza viral strains in addition to
the recombinant virus. In some embodiments, the vaccine comprises at least one influenza B
virus or influenza B virus antigen. In some embodiments, the vaccine comprises at least one
influenza C virus or influenza C virus antigen. In some embodiments, the vaccine comprises one
or more viruses or viral antigens comprising human influenza A and pandemic influenza viruses
from non-human species. In some embodiments, the vaccine comprises the human influenza A
virus is selected from the group comprising H1N1, H2N2 and H3N2.
[0016] Also described is a method for propagating a recombinant influenza virus, comprising:
contacting a host cell with a recombinant influenza virus SEQ ID NO:1, SEQ ID NO:2 or SEQ
ID NO: 3; incubating the host cell for a sufficient time and under conditions suitable for viral
replication, and isolating progeny virus particles.
[0017] Also described is a method of preparing a vaccine, comprising: placing a host cell in a
bioreactor; contacting the host cell with a recombinant virus SEQ ID NO:1, SEQ ID NO:2 or
SEQ ID NO: 3; incubating the host cell for a sufficient time and under conditions suitable for
viral propagation; isolating the progeny virus particles; and formulating the progeny virus
particles for administration as a vaccine.
[0018] Also described is a method for immunizing a subject, comprising: administering a
composition comprising a recombinant influenza virus comprising mutation in the M gene,
48204255.2
wherein the mutation in the M gene results in failure of the virus to express the M2 protein, or
causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID
NO:4.
[0019] Also described is a method for reducing the likelihood or severity of infection by
influenza A virus in a subject comprising: administering a composition comprising a
recombinant influenza virus comprising mutation in the M gene, wherein the mutation in the M
gene results in failure of the virus to express the M2 protein, or causes the virus to express a
truncated M2 protein having the amino acid sequence of SEQ ID NO:4. In some embodiments,
the recombinant influenza virus comprises SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In
some embodiments, the method comprises providing at least one booster dose of the
composition, wherein the at least one booster dose is provided at three weeks after a first
administration. In some embodiments, the method comprises administering the composition
intranasally, intramuscularly or intracutaneously. In some embodiments, the method comprises
administering is performed intracutaneously. In some embodiments, the method comprises
administering is performed using a microneedle delivery device.
[0020] Also described is a method for intracutaneous administration of an immunogenic
composition comprising: (a) providing a microneedle delivery device comprising (i) a puncture
mechanism; (ii) an immunogenic composition layer comprising a plurality of microneedles
capable of puncturing skin and allowing an immunogenic composition to be administered
intracutaneously; and (b) depressing the puncture mechanism; wherein the immunogenic
composition comprises a recombinant influenza virus comprising a mutation in the M gene, and
wherein the mutation in the M gene results in failure of the virus to express the M2 protein, or
causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID
NO:4. In some embodiments, the recombinant influenza virus comprises SEQ ID NO:1, SEQ ID
NO:2, or SEQ ID NO:3. In some embodiments, the microneedle array is initially positioned
inside of a device housing, and upon actuation of a lever allows the microneedles to extend
6
48204255.2
through the device bottom and insert into the skin thereby allowing infusion of the vaccine fluid
into the skin.
[0021] Also described is a recombinant influenza virus comprising a mutation in the M gene,
wherein the virus does not replicate in an unmodified host cell selected from the group consisting
of a Chinese Hamster Ovary (CHO) cell, a Vero cell, a or Madin-Darby canine kidney cell. In
some embodiments, the mutation in the M gene results in failure of the virus to express the M2
protein, or causes the virus to express a truncated M2 protein having the amino acid sequence of
SEQ ID NO:4.
[0022] Also described is a recombinant cell comprising a nucleic acid encoding an influenza
virus M2 ion channel gene, wherein the nucleic acid is expressed in the cell.
[0023 Also described is a recombinant cell comprising a 2,6-sialic acid receptor gene.
[0024 Also described is a recombinant cell comprising a cellular genome or an expression vector
that expresses (i) a viral M2 ion channel gene, and (ii) a 2,6-sialic acid receptor gene. In some
embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a
Chinese Hamster Ovary cell or a Vero cell. In some embodiments, the recombinant cell further
comprises a human influenza virus, wherein the virus does not express a functional M2 protein.
[0025] Also described is a method for producing recombinant influenza viral particles,
comprising (A) infecting the cell of one of claims 47-52 with human influenza virus, wherein the
cell either (i) constitutively expresses the functional M2 ion channel protein, or (ii) is induced
after viral infection to express the functional M2 ion channel protein, and wherein the virus
successfully replicates only in the presence of the functional M2 ion channel proteins expressed
by the cell; and (B) isolating the progeny virus particles. In some embodiments, the method
further comprises formulating the isolated viral particles into a vaccine. In some embodiments,
the virus comprises a human influenza virus, and wherein the virus does not express a functional
M2 protein.
7
48204255.2
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGURE 1 is a graphic depicting the role of M2 ion channel in an influenza virus life
cycle, wherein (1) the influenza virus attaches to sialic acid receptors on a cell surface; (2) the
virus is internalized into the cell; (3) the M2 ion channel is expressed on the viral surface; (4) the
M2 ion channel opens to permit proton entry, leading to a release of viral RNA that enters the
nucleus, is replicated and results in viral protein synthesis; and (5) the viral components are
packaged into virions and released.
[0027] FIGURE 2 is a schematic diagram of wild-type and mutant M2 genes. The M2 gene of
A/Puerto Rico/8/1934 (PR8) M segment was deleted by insertion of two stop codons
downstream of the open reading frame of the M1 protein followed by deletion of 51 nucleotides
in the transmembrane domain to inhibit expression of full-length M2 protein.
[0028] FIGURE 3 shows the nucleotide sequence (SEQ ID NO: 28) of unprocessed M1 and
M2.
[0029] FIGURE 4 is a chart showing the growth kinetics of M2KO(ΔTM) (upper panel) and
wild-type PR8 (lower panel) viruses in normal MDCK cells and MDCK cells stably expressing
M2 protein (M2CK). Cells were infected with viruses at multiplicity of infection of 10-5. Virus
titers in cell supernatants were determined. Wild-type PR8 grew to high titers in both cell types
whereas M2KO(ΔTM) grew well only in M2CK cells and not at all in MDCK cells.
[0030] FIGURE 5 is a western blot showing that M2KO(ΔTM) virus produces viral antigens,
but not M2, in normal cells. Cellular lysates were probed with PR8-infected mouse sera (panel
A) or anti-M2 monoclonal antibody (panel B). Lane 1, Molecular weight marker; Lane 2,
MDCK cells infected with PR8; Lane 3 MDCK cells infected with M2KO(ΔTM); Lane 4,
Uninfected MDCK cells.
8
48204255.2
[0031] FIGURE 6 is a chart showing the change in mouse body weight after inoculation with
M2KO variants.
[0032] FIGURE 7A is a chart showing antibody response in mice inoculated with M2KO
variants.
[0033] FIGURE 7B is a chart showing anti-PR8 IgG antibody titer in the serum of boosted
mice 6 weeks post infection.
[0034] FIGURE 8 is a chart showing change in mouse body weight after influenza challenge,
post-inoculation with M2KO variants.
[0035] FIGURE 9 is a chart showing mouse survival after influenza challenge, post-inoculation
with M2KO variants.
[0036] FIGURE 10 is a chart showing the change in mouse body weight after inoculation with
PR8 intranasally (IN), intradermally (ID) or intramuscularly (IM).
[0037] FIGURE 11A is a chart showing antibody titer in serum, collected at 2 weeks postinoculation with PR8, from mouse with 1.8x101 pfu (Lo) or 1.8x104 pfu (Hi) concentration of
virus. FIGURE 11B is a chart showing antibody titer in serum, collected at 7 weeks postinoculation with PR8, from mouse with 1.8x101 pfu (Lo) or 1.8x104 pfu (Hi) concentration of
vaccine.
[0038] FIGURE 12 is a chart showing mouse survival after influenza challenge, postinoculation with PR8.
[0039] FIGURE 13 is a chart showing change in mouse body weight after influenza challenge,
post-inoculation with PR8.
9
48204255.2
[0040] FIGURE 14 is a chart showing antibody titer in serum, collected from a mouse
inoculated with 1.8x104 pfu PR8 intradermally at 7 weeks post-inoculation.
[0041] FIGURE 15 is a chart showing the change in body weight of mice inoculated with
1.8x104 pfu PR8 intradermally.
[0042] FIGURE 16 is a chart showing % survival post challenge for mice infected with a
heterosubtypic virus.
[0043] FIGURE 17 is a chart showing ELISA titers of mice from different vaccination groups.
[0044] FIGURE 18 is a chart showing % survival of mice after homosubtypic virus infection.
[0045] FIGURE 19 is a chart showing % survival of mice after hetersubtypic virus challenge.
[0046] FIGURE 20 is a chart showing changes in body weight of inoculated ferrets. Ferrets
were inoculated with 107 TCID50 of M2KO(ΔTM) virus (panel A) or with 107 TCID50 of
A/Brisbane/10/2007 (H3N2) influenza A virus (panel B). Body weight was monitored for 3 days
post inoculation.
[0047] FIGURE 21 is a chart showing changes in body temperature of inoculated ferrets.
Ferrets were inoculated with 107 TCID50 of M2KO(ΔTM) virus (panel A) or with 107 TCID50 of
A/Brisbane/10/2007 (H3N2) influenza A virus (panel B). Body temperature was monitored for 3
days post inoculation.
[0048] FIGURE 22 is a chart showing changes in body weight of ferrets after vaccination.
Ferrets were inoculated with 107 TCID50 of M2KO(ΔTM) virus [G1 and G3], with 107 TCID50 of
FM#6 virus [G2 and G4] or OPTI-MEM™ [G5]. Changes in body weight were monitored for
14 days following prime vaccination (panel A) and after receiving a booster vaccine (panel B).
48204255.2
[0049] FIGURE 23 is a chart showing changes in body weight of ferrets after challenge.
Ferrets were challenged with 107 TCID50 of A/Brisbane/10/2007 (H3N2) influenza A virus.
Body weight was monitored for 14 days post inoculation.
[0050] FIGURE 24 is a chart showing changes in body temperature of ferrets after vaccination.
Ferrets were inoculated with 107 TCID50 of M2KO(ΔTM) virus [G1 and G3], with 107 TCID50 of
FM#6 virus [G2 and G4] or OPTI-MEM™ [G5]. Changes in body temperature were monitored
for 14 days following prime vaccination (panel A) and after receiving a booster vaccine (panel
B).
[0051] FIGURE 25 is a chart showing changes in body tempertature of ferrets after challenge.
Ferrets were challenged with 107 TCID50 of A/Brisbane/10/2007 (H3N2) influenza A virus.
Body temperature was monitored for 14 days post inoculation.
[0052] FIGURE 26 is a chart showing changes in weight of ferrets after virus inoculation.
Donor ferrets were inoculated on day 0 with either 10 7 TCID50 of M2KO(ΔTM) virus (panel A)
or with 107 TCIDso of A/Brisbane/10/2007 (H3N2) virus (panel B). 24 hours (Day 1) after
inoculation donors were placed in a cage with direct contacts (DC) adjacent to a cage housing an
aerosol contact (AC). Changes in body weight were monitored for 14 days following donor
inoculation.
[0053] FIGURE 27 is a chart showing changes in body temperature of ferrets after virus
inoculation. Donor ferrets were inoculated on day 0 with either 107 TCID50 ofM2KO(ΔTM)
virus (panel A) or with 107 TCID50 of A/Brisbane/10/2007 (H3N2) virus (panel B). 24 hours
(Day 1) after inoculation donors were placed in a cage with direct contacts (DC) adjacent to a
cage housing an aerosol contact (AC). Changes in body temperature were monitored for 14 days
following donor inoculation.
[0054] FIGURE 28 is a chart showing that M2KO(ΔTM) vaccine elicits humoral and mucosal
responses. Panel A shows serum IgG and IgA titers following administration of PR8,
11
48204255.2
M2KO(ΔTM), inactivated PR8 (IN, IM), or PBS. Panel B shows lung wash IgG and IgA titers
following administration of PR8, M2KO(ΔTM), inactivated PR8 (IN, IM), or PBS.
[0055] FIGURE 29 is a chart showing that M2KO(ΔTM) vaccine protects mice from lethal
homosubtypic and heterosubtypic viral challenge. Panel A shows mouse body weight change
following homologous PR8 (H1N1) challenge. Panel B shows mouse survival following
heterologous Aichi (H3N2) challenge.
[0056] FIGURE 30 is a chart showing that M2KO(ΔTM) vaccine controls challenge virus
replication in respiratory tract. Panel A shows viral titers following PR8 (H1N1) challenge.
Panel B shows viral titers following Aichi (H3N2) challenge.
[0057] FIGURE 31 is a chart showing the kinetics of antibody response to M2KO(ΔTM)
vaccine in sera.
[0058] FIGURE 32 is a chart showing the mucosal antibody response to M2KO(ΔTM) vaccine
in sera and respiratory tract.
[0059] FIGURE 33 is a chart showing the kinetics of anti-HA IgG in mice in response to
M2KO(ΔTM) vaccine.
[0060] FIGURE 34 is a chart showing that M2KO(ΔTM) vaccine induces immune responses
similar to FluMist® and IVR-147. Panel A shows serum viral titers in animals administered
FluMist® H3, M2KO(ΔTM) H3, IVR-147, and PBS. Panel B shows lung wash viral titers in
animals administered FluMist® H3, M2KO(ΔTM) H3, IVR-147, and PBS. Panel C shows nasal
turbinate viral titiers in animals administered FluMist® H3, M2KO(ΔTM) H3, IVR-147, and
PBS.
[0061] FIGURE 35 is a chart showing that M2KO(ΔTM) vaccine protects against Aichi
challenge. Panel A shows body weight loss following Aichi challenge in animals administered
FluMist® H3, M2KO(ΔTM) H3, IVR-147, and PBS. Panel B shows the percent survival
12
48204255.2
following Aichi challenge of animals administered FluMist® H3, M2KO(ΔTM) H3, IVR-147,
and PBS.
[0062] FIGURE 36 is a chart showing that H5N1 M2KO(ΔTM) vaccine elicits IgG antibody
titers against HA.
[0063] FIGURE 37 is a chart showing body weight following administration of M2KO(ΔTM)
CA07, WT CA07, and FluMist® CA07 vaccines.
[0064] FIGURE 38 is a chart showing that M2KO(ΔTM) virus does not replicate in respiratory
tract of mice.
[0065] FIGURE 39 is a chart showing that M2KO(ΔTM) vaccine displays rapid antibody
kinetics.
[0066] FIGURE 40 is a chart showing that M2KO(ΔTM) vaccine protects against heterologous
challenge with H3N2 virus, A/Aichi/2/1968.
[0067] FIGURE 41 is a chart showing that M2KO(ΔTM) vaccine primes for cellular responses
that are recalled upon challenge.
[0068] FIGURE 42 is a chart showing that M2KO(ΔTM) virus generates mRNA levels similar
to virus wild-type for M2.
[0069] FIGURE 43 is an agarose gel showing restriction digests of the pCMV-PR8-M2
expression plasmid. Lanes 1 & 5; 1 Kb DNA Ladder (Promega,Madison, WI, USA), Lane 2-4;
Eco R1 digested pCMLV-PR8-M2: 0.375 µg (Lane 2), 0.75 µg (Lane 3), and 1.5 µg (Lane 4).
[0070] FIGURE 44 is a chart showing a sequence alignment of pCMV –PR8-M2 to the open
reading frame of the influenza M2 gene. FIGURE 44 discloses SEQ ID NOS 29-33, respectively,
in order of appearance.
13
48204255.2
[0071] FIGURE 45 is a chart showing M2KO(ΔTM) and FluMist® virus replication in the
ferret respiratory tract.
[0072] FIGURE 46 is a chart showing M2KO(ΔTM) and FluMist® viral titers in nasal washes
after intranasal challenge with A/Brisbane/10/2007 (H3N2) virus.
[0073] FIGURE 47 is a chart showing IgG titers in ferrets following vaccination with
M2KO(ΔTM) and FluMist,® prime group only.
[0074] FIGURE 48 is a chart showing IgG titers in ferrets following vaccination with
M2KO(ΔTM) and FluMist,® prime-boost groups.
[0075] FIGURE 49 is a chart showing a summary of ELISA IgG titers in ferret sera from
vaccination with M2KO(ΔTM) or FluMist® to post-challenge.
[0076] FIGURE 50 is a chart showing viral titers in nasal washes from ferrets in transmission
study. M2KO(ΔTM) virus did not transmit (no virus detected), whereas the control Brisb/10
virus did transmit.[0077] FIGURE 51 is a chart showing IgG titers in subjects vaccinated with
A/California, A/Perth, and B/Brisbane viruses intranasally (IN), intramuscularly (IM) and
intradermally (ID FGN).
[0078] FIGURE 52 is a chart showing IgG titers in subjects administered a priming does or a
priming and booster dose of A/Perth (H3N2) vaccine intramuscularly (IM) or intradermally (ID
FGN).
[0079] FIGURE 53 is a chart showing viral titers in guinea pigs inoculated with FluLaval:
A/California/7/2009 NYMC X-181, A/Victoria/210/2009 NYMC X-187 (an A/Perth/16/2009-
like virus), and B/Brisbane/60/2008 by intramuscular (IM) and intradermal (ID) delivery at 0, 30,
and 60 days post-inoculation.
14
48204255.2
[0080] FIGURE 54 is a chart showing the percent survival of H5N1 M2KO(ΔTM) vaccinated
subjects challenged 5 months post-immunization with Vietnam/1203/2004 virus.
[0081] FIGURE 55 is a chart showing the percent survival of H5N1 M2KO(ΔTM) vaccinated
subjects challenged 4 weeks post-immunization with Vietnam/1203/2004 virus.
DETAILED DESCRIPTION
I. Definitions
[0082] The following terms are used herein, the definitions of which are provided for guidance.
[0083] As used herein, the singular forms “a,” “an,” and “the” designate both the singular and
the plural, unless expressly stated to designate the singular only.
[0084] The term “about” and the use of ranges in general, whether or not qualified by the term
about, means that the number comprehended is not limited to the exact number set forth herein,
and is intended to refer to ranges substantially within the quoted range while not departing from
the scope of the invention. As used herein, “about” will be understood by persons of ordinary
skill in the art and will vary to some extent on the context in which it is used. If there are uses of
the term which are not clear to persons of ordinary skill in the art given the context in which it is
used, “about” will mean up to plus or minus 10% of the particular term.
[0085] As used herein “subject” and “patient” are used interchangeably and refer to an animal,
for example, a member of any vertebrate species. The methods and compositions of the presently
disclosed subject matter are particularly useful for warm-blooded vertebrates including mammals
and birds. Exemplary subjects may include mammals such as humans, as well as mammals and
birds of importance due to being endangered, of economic importance (animals raised on farms
for consumption by humans) and/or of social importance (animals kept as pets or in zoos) to
humans. In some embodiments, the subject is a human. In some embodiments, the subject is not
human.
48204255.2
[0086] As used herein, the terms “effective amount” or “therapeutically effective amount” or
“pharmaceutically effective amount” refer to a quantity sufficient to achieve a desired
therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, disease,
condition and/or symptom(s) thereof. In the context of therapeutic or prophylactic applications,
the amount of a composition administered to the subject will depend on the type and severity of
the disease and on the characteristics of the individual, such as general health, age, sex, body
weight and tolerance to the composition drugs. It will also depend on the degree, severity and
type of disease or condition. The skilled artisan will be able to determine appropriate dosages
depending on these and other factors. In some embodiments, multiple doses are administered.
Additionally or alternatively, in some embodiments, multiple therapeutic compositions or
compounds (e.g., immunogenic compositions, such as vaccines) are administered.
[0087] As used herein, the terms “isolated” and/or “purified” refer to in vitro preparation,
isolation and/or purification of a nucleic acid (e.g., a vector or plasmid), polypeptide, virus or
cell such that it is not associated with unwanted in vivo substances, or is substantially purified
from unwanted in vivo substances with which it normally occurs. For example, in some
embodiments, an isolated virus preparation is obtained by in vitro culture and propagation, and is
substantially free from other infectious agents. As used herein, “substantially free” means below
the level of detection for a particular compound, such as unwanted nucleic acids, proteins, cells,
viruses, infectious agents, etc. using standard detection methods for that compound or agent.
[0088] As used herein the term “recombinant virus” refers to a virus that has been manipulated
in vitro, e.g., using recombinant nucleic acid techniques, to introduce changes to the viral
genome and/or to introduce changes to the viral proteins. For example, in some embodiments,
recombinant viruses may include both wild-type, endogenous, nucleic acid sequences and mutant
and/or exogenous nucleic acid sequences. Additionally or alternatively, in some embodiments,
recombinant viruses may include modified protein components, such as mutant or variant matrix,
hemagglutinin, neuraminidase, nucleoprotein, non-structural and/or polymerase proteins.
16
48204255.2
[0089] As used herein the term “recombinant cell” or “modified cell” refer to a cell that has
been manipulated in vitro, e.g., using recombinant nucleic acid techniques, to introduce nucleic
acid into the cell and/or to modify cellular nucleic acids. Examples of recombinant cells includes
prokaryotic or eukaryotic cells carrying exogenous plasmids, expression vectors and the like,
and/or cells which include modifications to their cellular nucleic acid (e.g., substitutions,
mutations, insertions, deletions, etc., into the cellular genome). An exemplary recombinant cell
is one which has been manipulated in vitro to express an exogenous protein, such as a viral M2
protein.
[0090] As used herein the terms “mutant,” “mutation,” and “variant” are used interchangeably
and refer to a nucleic acid or polypeptide sequence which differs from a wild-type sequences. In
some embodiments, mutant or variant sequences are naturally occurring. In other embodiments,
mutant or variant sequence are recombinantly and/or chemically introduced. In some
embodiments, nucleic acid mutations include modifications (e.g., additions, deletions,
substitutions) to RNA and/or DNA sequences. In some embodiments, modifications include
chemical modification (e.g., methylation) and may also include the substitution or addition of
natural and/or non-natural nucleotides. Nucleic acid mutations may be silent mutations (e.g., one
or more nucleic acid changes which code for the same amino acid as the wild-type sequence) or
may result in a change in the encoded amino acid, result in a stop codon, or may introduce
splicing defects or splicing alterations. Nucleic acid mutations to coding sequences may also
result in conservative or non-conservative amino acid changes.
[0091] As used herein, the term “vRNA” refers to the RNA comprising a viral genome,
including segmented or non-segmented viral genomes, as well as positive and negative strand
viral genomes. vRNA may be wholly endogenous and “wild-type” and/or may include
recombinant and/or mutant sequences.
[0092] As used herein, the term “host cell” refers to a cell in which a pathogen, such as a virus,
can replicate. In some embodiments, host cells are in vitro, cultured cells (e.g., CHO cells, Vero
17
48204255.2
cells, MDCK cells, etc.) Additionally or alternatively, in some embodiments, host cells are in
vivo (e.g., cells of an infected vertebrate, such as an avian or mammal). In some embodiments,
the host cells may be modified, e.g., to enhance viral production such as by enhancing viral
infection of the host cell and/or by enhancing viral growth rate. By way of example, but not by
way of limitation, exemplary host cell modifications include recombinant expression of 2
linked sialic acid receptors on the cell surface of the host cell, and/or recombinant expression of
a protein in the host cells that has been rendered absent or ineffective in the pathogen or virus.
[0093] As used herein, the term “infected” refers to harboring a disease or pathogen, such as a
virus. An infection can be intentional, such as by administration of a virus or pathogen (e.g., by
vaccination), or unintentional, such as by natural transfer of the pathogen from one organism to
another, or from a contaminated surface to the organism.
[0094] As used herein, the term “attenuated,” as used in conjunction with a virus, refers to a
virus having reduced virulence or pathogenicity as compared to a non-attenuated counterpart, yet
is still viable or live. Typically, attenuation renders an infectious agent, such as a virus, less
harmful or virulent to an infected subject compared to a non-attenuated virus. This is in contrast
to killed or completely inactivated virus.
[0095] As used herein, the term “type” and “strain” as used in conjunction with a virus are
used interchangeably, and are used to generally refer to viruses having different characteristics.
For example, influenza A virus is a different type of virus than influenza B virus. Likewise,
influenza A H1N1 is a different type of virus than influenza A H2N1, H2N2 and H3N2.
Additionally or alternatively, in some embodiments, different types of virus such as influenza A
H2N1, H2N2 and H3N2 may be termed “subtypes.”
[0096] As used herein, “M2KO” or “M2KO(ΔTM)” refers to SEQ ID NO:1, a virus
comprising SEQ ID NO:1, or a vaccine comprising a virus comprising SEQ ID NO:1, depending
on the context in which it is used. For example, in describing mutations of the M2 gene
18
48204255.2
demonstrated herein, “M2KO” or “M2KO(ΔTM)” refers to SEQ ID NO:1. When describing the
viral component of a vaccine, “M2KO” or “M2KO(ΔTM)” refers to a recombinant influenza
virus which possesses internal 6 genes of PR8 (nucleoprotein (NP), polymerase genes (PA, PB1,
PB2), non-structural (NS), matrix (M)), but which does not express functional M2 protein.
When describing a vaccine, “M2KO” or “M2KO(ΔTM)” refers to a vaccine comprising the
M2KO(ΔTM) recombinant virus.
[0097] As used herein , “M2KO(ΔTM) virus” encompasses a recombinant influenza virus
which possesses internal 6 genes of PR8 (nucleoprotein (NP), polymerase genes (PA, PB1, PB2),
non-structural (NS), matrix (M)), but which does not express functional M2 protein, alone or in
combination with other viral components and/or genes encoding other viral components. In
some embodiments, the M2KO(ΔTM) virus comprises genes of other influenza viruses. In some
embodiments, the virus comprises the HA and NA genes of Influenza A/Brisbane/10/2007-like
A/Uruguay/716/2007(H3N2). In some embodiments, the M2KO(ΔTM) virus comprises the HA
and NA genes of the A/Vietnam/1203/2004 (H5N1) virus. In some embodiments, the
M2KO(ΔTM) virus comprises the HA and NA genes of the A/California/07/2009 (CA07)
(H1N1pdm) virus.
II. Influenza A virus
A. General
[0098] Influenza is a leading cause of death among American adults. The causal agent of
influenza are viruses of the family orthomyxoviridae including influenza A virus, influenza B
virus and influenza C virus, with influenza A being the most common and most virulent in
humans.
[0099] The influenza A virus is an enveloped, negative-strand RNA virus. The genome of
influenza A virus is contained on eight single (non-paired) RNA strands the complements of
which code for eleven proteins (HA, NA, NP, M1, M2, NS1, NEP, PA, PB1, PB1-F2, PB2). The
19
48204255.2
total genome size is about 14,000 bases. The segmented nature of the genome allows for the
exchange of entire genes between different viral strains during cellular cohabitation. The eight
RNA segments are as follows. 1) HA encodes hemagglutinin (about 500 molecules of
hemagglutinin are needed to make one virion); 2) NA encodes neuraminidase (about 100
molecules of neuraminidase are needed to make one virion); 3) NP encodes nucleoprotein; 4) M
encodes two proteins (the M1 and the M2) by using different reading frames from the same RNA
segment (about 3000 M1 molecules are needed to make one virion); 5) NS encodes two proteins
(NS1 and NEP) by using different reading frames from the same RNA segment; 6) PA encodes
an RNA polymerase; 7) PB1 encodes an RNA polymerase and PB1-F2 protein (induces
apoptosis) by using different reading frames from the same RNA segment; 8) PB2 encodes an
RNA polymerase.
[0100] There are several subtypes of influenza A, named according to an H number (for the
type of hemagglutinin) and an N number (for the type of neuraminidase). Currently, there are 16
different H antigens known (H1 to H16) and nine different N antigens known (N1 to N9). Each
virus subtype has mutated into a variety of strains with differing pathogenic profiles; some
pathogenic to one species but not others, some pathogenic to multiple species. Exemplary
Influenza A virus subtypes that have been confirmed in humans, include, but are not limited to
H1N1 which caused the “Spanish Flu” and the 2009 swine flu outbreak; H2N2 which caused the
“Asian Flu” in the late 1950s; H3N2 which caused the Hong Kong Flu in the late 1960s; H5N1,
considered a global influenza pandemic threat through its spread in the mid-2000s; H7N7; H1N2
which is currently endemic in humans and pigs; and H9N2, H7N2, H7N3, H5N2, H10N7.
[0101] Some influenza A variants are identified and named according to the known isolate to
which they are most similar, and thus are presumed to share lineage (e.g., Fujian flu virus-like);
according to their typical host (example Human flu virus); according to their subtype (example
H3N2); and according to their pathogenicity (example LP, Low Pathogenic). Thus, a flu from a
48204255.2
virus similar to the isolate A/Fujian/411/2002(H3N2) can be called Fujian flu, human flu, and
H3N2 flu.
[0102] In addition, influenza variants are sometimes named according to the species (host) the
strain is endemic in or adapted to. The main variants named using this convention are: bird flu,
human flu, swine influenza, equine influenza and canine influenza. Variants have also been
named according to their pathogenicity in poultry, especially chickens, e.g., Low Pathogenic
Avian Influenza (LPAI) and Highly Pathogenic Avian Influenza (HPAI).
B. Life cycle and structure
[0103] The life cycle of influenza viruses generally involves attachment to cell surface
receptors, entry into the cell and uncoating of the viral nucleic acid, followed by replication of
the viral genes inside the cell. After the synthesis of new copies of viral proteins and genes, these
components assemble into progeny virus particles, which then exit the cell. Different viral
proteins play a role in each of these steps.
[0104] The influenza A particle is made up of a lipid envelope which encapsulates the viral
core. The inner side of the envelope is lined by the matrix protein (M1), while the outer surface
is characterized by two types of glycoprotein spikes: hemagglutinin (HA) and neuraminidase
(NA). M2, a transmembrane ion channel protein, is also part of the lipid envelope. See e.g.,
Figure 1.
[0105] The HA protein, a trimeric type I membrane protein, is responsible for binding to
sialyloligosaccharides (oligosaccharides containing terminal sialic acid linked to galactose) on
host cell surface glycoproteins or glycolipids. This protein is also responsible for fusion between
viral and host cell membranes, following virion internalization by endocytosis.
[0106] Neuraminidase (NA), a tetrameric type II membrane protein, is a sialidase that cleaves
terminal sialic acid residues from the glycoconjugates of host cells and the HA and NA, and thus
21
48204255.2
is recognized as receptor-destroying enzyme. This sialidase activity is necessary for efficient
release of progeny virions from the host cell surface, as well as prevention of progeny
aggregation due to the binding activity of viral HAs with other glycoproteins. Thus, the
receptor-binding activity of the HA and the receptor-destroying activity of the NA likely act as
counterbalances, allowing efficient replication of influenza.
[0107] The genome segments are packaged into the core of the viral particle. The RNP (RNA
plus nucleoprotein, NP) is in helical form with three viral polymerase polypeptides associated
with each segment.
[0108] The influenza virus life cycle begins with binding of the HA to sialic acid-containing
receptors on the surface of the host cell, followed by receptor-mediated endocytosis. Figure 1.
The low pH in late endosomes triggers a conformational shift in the HA, thereby exposing the Nterminus of the HA2 subunit (the so-called fusion peptide). The fusion peptide initiates the
fusion of the viral and endosomal membrane, and the matrix protein (M1) and RNP complexes
are released into the cytoplasm. RNPs consist of the nucleoprotein (NP), which encapsidates
vRNA, and the viral polymerase complex, which is formed by the PA, PB1, and PB2 proteins.
RNPs are transported into the nucleus, where transcription and replication take place. The RNA
polymerase complex catalyzes three different reactions: (1) synthesis of an mRNA with a 5' cap
and 3' polyA structure, (2) a full-length complementary RNA (cRNA), and (3) genomic vRNA
using the cDNA as a template. Newly synthesized vRNAs, NP, and polymerase proteins are
then assembled into RNPs, exported from the nucleus, and transported to the plasma membrane,
where budding of progeny virus particles occurs. The neuramimidase (NA) protein plays a role
late in infection by removing sialic acid from sialyloligosaccharides, thus releasing newly
assembled virions from the cell surface and preventing the self aggregation of virus particles.
Although virus assembly involves protein-protein and protein-vRNA interactions, the nature of
these interactions remains largely unknown.
22
48204255.2
C. Role of the M2 protein
[0109] As described above, spanning the viral membrane are three proteins: hemagglutinin
(HA), neuramimidase (NA), and M2. The extracellular domains (ectodomains) of HA and NA
are quite variable, while the ectodomain domain of M2 is essentially invariant among influenza
A viruses. Without wishing to be bound by theory, in influenza A viruses, the M2 protein which
possesses ion channel activity, is thought to function at an early state in the viral life cycle
between host cell penetration and uncoating of viral RNA. Once virions have undergone
endocytosis, the virion-associated M2 ion channel, a homotetrameric helix bundle, is believed to
permit protons to flow from the endosome into the virion interior to disrupt acid-labile M1
protein-ribonucleoprotein complex (RNP) interactions, thereby promoting RNP release into the
cytoplasm. In addition, among some influenza strains whose HAs are cleaved intracellularly
(e.g., A/fowl plagues/Rostock/34), the M2 ion channel is thought to raise the pH of the transGolgi network, preventing conformational changes in the HA due to conditions of low pH in this
compartment. It was also shown that the M2 transmembrane domain itself can function as an ion
channel. M2 protein ion channel activity is thought to be essential in the life cycle of influenza
viruses, because amantadine hydrochloride, which blocks M2 ion channel activity, has been
shown to inhibit viral replication. However, a requirement for this activity in the replication of
influenza A viruses has not been directly demonstrated. The structure of the M2 protein is
shown in Figure 2. The nucleic acid sequence of the M2 protein, along with the M1 sequence, is
shown in Figure 3.
[0110] Although influenza B and C viruses are structurally and functionally similar to
influenza A virus, there are some differences. For example, influenza B virus does not have an
M2 protein with ion channel activity. Instead, the NB protein, a product of the NA gene, likely
has ion channel activity and thus a similar function to the influenza A virus M2 protein.
Similarly, influenza C virus does not have an M2 protein with ion channel activity. However, the
CM1 protein of the influenza C virus is likely to have this activity.
23
48204255.2
III. M2 viral mutants
[0111] In one aspect, influenza A viruses harboring a mutant M2 vRNA sequence are
disclosed. Typically, such mutants do not have M2 ion channel activity, exhibit attenuated
growth properties in vivo, cannot produce infectious progeny and are non-pathogenic or show
reduced pathogenesis in infected subjects. The mutant viruses are immunogenic, and when used
as a vaccine, provide protection against infection with a counterpart wild-type and/or other
pathogenic virus. Additionally, the M2 mutants disclosed herein are stable, and do not mutate to
express a functional M2 polypeptide, regardless of the host cell used. Additionally or
alternatively, in some embodiments, the M1 protein of these mutants is produced without
detectable alteration to its function. In some embodiments, viruses harboring the mutant M2
nucleic acid sequences can not replicate in a host cell in which a corresponding wild-type virus
could be propagated. By way of example, but not by way of limitation, in some embodiments,
the wild-type virus can be grown, propagated and replicate in culturing MDCK cells, CHO cells
and/or Vero cells, while the corresponding virus harboring a mutant M2 sequence cannot grow,
replicate or be propagated in the same type of cells.
[0112] As noted above, in some embodiments, the M2 mutant virus is stable, and does not
mutate or revert to wild-type or to a non-wild-type sequence encoding a functional M2 protein in
a host cell. For example, in some embodiments, the M2 mutant virus is stable for 2 passages, 3
passages, 5 passages, 10 passages, 12 passages, 15 passages, 20 passages, 25 passages or more
than 25 passages in a host cell. In some embodiments, the host cell is an unmodified host cell.
In other embodiments, the host cell is a modified host cell, such as a MDCK cell which
expresses the M2 protein.
[0113] In some embodiments, the M2 mutants include one or more nucleic acid substitutions
and/or deletions. In some embodiments, the mutations are localized in nucleic acids which code
for one or more of the extracellular domain of the M2 protein, the transmembrane domain of the
M2 proteins and/or the cytoplasmic tail of the M2 protein. Additionally or alternatively, in some
24
48204255.2
embodiments, one or more nucleic acid mutations results in a splice variant, one or more stop
codons and/or one or more amino acid deletions of the M2 peptide In some embodiments,
viruses carrying the mutant M2 nucleic acid produce a non-functional M2 polypeptide. In some
embodiments, viruses carrying the mutant M2 nucleic acid do not produce an M2 polypeptide.
In some embodiments, viruses carrying the mutant M2 nucleic acid produce a truncated M2
polypeptide. In some embodiments, truncated M2 polypeptide has the amino acid sequence
MSLLTEVETPIRNEWGCRCNGSSD (SEQ ID NO: 4).
[0114] Three exemplary, non-limiting M2 viral mutants (M2-1, M2-2 and M2-3) are provided
below in Tables 1-3. In the tables, lower case letters correspond to the M2 sequence; upper case
letters correspond to the M1 sequence; mutant sequence (e.g., stop codons, splice defect) are in
bold, underlined. Underlined (lower case) bases in the M2-2 mutant indicate the region deleted
in the M2-1 and M2-3 mutants.
TABLE 1: M2-1 –(SEQ ID NO: 1) M2 ectodomain + 2 stop codons + TM deletion (PR8 M
segment + 2 stops (786-791) without 792-842 (TM)); also known as “M2KOTMdel,”
“M2KOΔTM.”
3’AGCAAAAGCAGGTAGATATTGAAAGatgagtcttctaaccgaggtcgaaacGTACGTACTCTCTA
TCATCCCGTCAGGCCCCCTCAAAGCCGAGATCGCACAGAGACTTGAAGATGTCTTTG
CAGGGAAGAACACCGATCTTGAGGTTCTCATGGAATGGCTAAAGACAAGACCAATC
CTGTCACCTCTGACTAAGGGGATTTTAGGATTTGTGTTCACGCTCACCGTGCCCAGT
GAGCGAGGACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTTAATGGGAACGGGGA
TCCAAATAACATGGACAAAGCAGTTAAACTGTATAGGAAGCTCAAGAGGGAGATAA
CATTCCATGGGGCCAAAGAAATCTCACTCAGTTATTCTGCTGGTGCACTTGCCAGTT
GTATGGGCCTCATATACAACAGGATGGGGGCTGTGACCACTGAAGTGGCATTTGGC
CTGGTATGTGCAACCTGTGAACAGATTGCTGACTCCCAGCATCGGTCTCATAGGCAA
ATGGTGACAACAACCAATCCACTAATCAGACATGAGAACAGAATGGTTTTAGCCAG
CACTACAGCTAAGGCTATGGAGCAAATGGCTGGATCGAGTGAGCAAGCAGCAGAGG
CCATGGAGGTTGCTAGTCAGGCTAGACAAATGGTGCAAGCGATGAGAACCATTGGG
ACTCATCCTAGCTCCAGTGCTGGTCTGAAAAATGATCTTCTTGAAAATTTGCAGgcctat
cagaaacgaatgggggtgcagatgcaacggttcaagtgatTAATAGgatcgtctttttttcaaatgcatttaccgtcgctttaaatacgg
actgaaaggagggccttctacggaaggagtgccaaagtctatgagggaagaatatcgaaaggaacagcagagtgctgtggatgctgacg
atggtcattttgtcagcatagagctggagtaaAAAACTACCTTGTTTCTACT
48204255.2
[0115] The M2 polypeptide sequence produced from this mutant is as follows:
MSLLTEVETPIRNEWGCRCNGSSD. (SEQ ID NO: 4).
TABLE 2: M2-2 – SEQ ID NO: 2 M2 ectodomain + 2 stops + splice defect (PR8 M segment + 2
stops (786-791) +splice defect nt 52) (also known as “Splice def M2KO” or “Splice def”)
3’AGCAAAAGCAGGTAGATATTGAAAGatgagtcttctaaccgaggtcgaaacCTACGTACTCTCTA
TCATCCCGTCAGGCCCCCTCAAAGCCGAGATCGCACAGAGACTTGAAGATGTCTTTG
CAGGGAAGAACACCGATCTTGAGGTTCTCATGGAATGGCTAAAGACAAGACCAATC
CTGTCACCTCTGACTAAGGGGATTTTAGGATTTGTGTTCACGCTCACCGTGCCCAGT
GAGCGAGGACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTTAATGGGAACGGGGA
TCCAAATAACATGGACAAAGCAGTTAAACTGTATAGGAAGCTCAAGAGGGAGATAA
CATTCCATGGGGCCAAAGAAATCTCACTCAGTTATTCTGCTGGTGCACTTGCCAGTT
GTATGGGCCTCATATACAACAGGATGGGGGCTGTGACCACTGAAGTGGCATTTGGC
CTGGTATGTGCAACCTGTGAACAGATTGCTGACTCCCAGCATCGGTCTCATAGGCAA
ATGGTGACAACAACCAATCCACTAATCAGACATGAGAACAGAATGGTTTTAGCCAG
CACTACAGCTAAGGCTATGGAGCAAATGGCTGGATCGAGTGAGCAAGCAGCAGAGG
CCATGGAGGTTGCTAGTCAGGCTAGACAAATGGTGCAAGCGATGAGAACCATTGGG
ACTCATCCTAGCTCCAGTGCTGGTCTGAAAAATGATCTTCTTGAAAATTTGCAGgcctat
cagaaacgaatgggggtgcagatgcaacggttcaagtgatTAATAGactattgccgcaaatatcattgggatcttgcacttgacattgt
ggattcttgatcgtctttttttcaaatgcatttaccgtcgctttaaatacggactgaaaggagggccttctacggaaggagtgccaaagtctatga
gggaagaatatcgaaaggaacagcagagtgctgtggatgctgacgatggtcattttgtcagcatagagctggagtaaAAAACTAC
CTTGTTTCTACT
[0116] No M2 polypeptide sequence is produced from this mutant.
TABLE 3: M2-3 – SEQ ID NO: 3 M2 ectodomain + 2 stops + splice defect + TM deletion (PR8
M segment + 2 stops (786-791) without 792-842 (TM)+splice defect nt 52) (also known as
TMdel + Splice def M2KO)
3’AGCAAAAGCAGGTAGATATTGAAAGatgagtcttctaaccgaggtcgaaacCTACGTACTCTCTA
TCATCCCGTCAGGCCCCCTCAAAGCCGAGATCGCACAGAGACTTGAAGATGTCTTTG
CAGGGAAGAACACCGATCTTGAGGTTCTCATGGAATGGCTAAAGACAAGACCAATC
CTGTCACCTCTGACTAAGGGGATTTTAGGATTTGTGTTCACGCTCACCGTGCCCAGT
GAGCGAGGACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTTAATGGGAACGGGGA
TCCAAATAACATGGACAAAGCAGTTAAACTGTATAGGAAGCTCAAGAGGGAGATAA
CATTCCATGGGGCCAAAGAAATCTCACTCAGTTATTCTGCTGGTGCACTTGCCAGTT
26
48204255.2
GTATGGGCCTCATATACAACAGGATGGGGGCTGTGACCACTGAAGTGGCATTTGGC
CTGGTATGTGCAACCTGTGAACAGATTGCTGACTCCCAGCATCGGTCTCATAGGCAA
ATGGTGACAACAACCAATCCACTAATCAGACATGAGAACAGAATGGTTTTAGCCAG
CACTACAGCTAAGGCTATGGAGCAAATGGCTGGATCGAGTGAGCAAGCAGCAGAGG
CCATGGAGGTTGCTAGTCAGGCTAGACAAATGGTGCAAGCGATGAGAACCATTGGG
ACTCATCCTAGCTCCAGTGCTGGTCTGAAAAATGATCTTCTTGAAAATTTGCAGgcctat
cagaaacgaatgggggtgcagatgcaacggttcaagtgatTAATAGgatcgtctttttttcaaatgcatttaccgtcgctttaaatacgg
actgaaaggagggccttctacggaaggagtgccaaagtctatgagggaagaatatcgaaaggaacagcagagtgctgtggatgctgacg
atggtcattttgtcagcatagagctggagtaaAAAACTACCTTGTTTCTACT
[0117] No M2 polypeptide sequence is produced from this mutant.
[0118] Additionally or alternatively, in some embodiments, M2 mutations are introduced into
the cytoplasmic tail. Figure 2. The M2 protein cytoplasmic tail is a mediator of infectious virus
production. In some embodiments, truncations of the M2 cytoplasmic tail result in a decrease in
infectious virus titers, a reduction in the amount of packaged viral RNA, a decrease in budding
events, and a reduction in budding efficiency. It has been shown that the 5’ sequence is more
important than 3’ sequence for genome packaging, and that a longer 5’ sequence is better for
genome packaging. In addition, studies have shown that nucleotide length is important, but the
actual sequence is less so (random sequences are sufficient to generate viruses). Stable M2
cytoplasmic tail mutants have been challenging to develop, and the literature includes numerous
examples of mutant reversion.
[0119] For example, Pekosz et al JVI, 2005; 79(6): 3595-3605, replaced two codons with stop
codons at amino acid position 70, but the virus soon reverted. Another exemplary M2
cytoplasmic tail mutation is termed M2del11. In the M2del11 mutant, 11 amino acid residues
are deleted from carboxyl end of cytoplasmic tail. This truncation is due to the introduction of
two stop codons, and a full length M2 polypeptide is not made. While this mutant is stable when
passaged in M2 expressing MDCK cells (M2CK), it reverts to full length M2 during passaging in
27
48204255.2
normal MDCK cells (J Virol. 2008 82(5):2486-92). Without wishing to be bound by theory, it is
likely that reversion occurs with selective pressure in the MDCK cells.
[0120] Another M2 cytoplasmic tail mutant, M2Stop90ala78-81 did not reduce virus titer but
ala70-77 did (JVI 2006; 80 (16) p8178-8189). Alanine-scanning experiments further indicated
that amino acids at positions 74 to 79 of the M2 tail play a role in virion morphogenesis and
affect viral infectivity. (J Virol. 2006 80(11):5233-40.)
[0121] Accordingly, presented herein are novel cytoplasmic mutants, with characteristics
different than those described above. For example, in some embodiments, the cytoplasmic
mutants are stable (do not revert to express a full-length M2 polypeptide) in MDCK cells. In
some embodiments, the cytoplasmic mutants are stable for 2 passages, 3 passages, 5 passages, 10
passages, 15 passages, 20 passages, 25 passages or more than 25 passages in a host cell.
[0122] The wild-type M2 polypeptide is shown below in Table 4. For each of the sequences,
the bold text indicates the transmembrane domain. The extracellular domain is first (left),
followed by the transmembrane domain (center) and the cytoplasmic tail sequence (right).
TABLE 4: Wild-type M2 polypeptide and cytoplasmic tail mutants
Wild-type M2 polypeptide
MSLLTEVETPIRNEWGCRCNGSSDPLTIAANIIGILHLTLWILDRLFFKCIYRRFKYGLK
GGPSTEGVPKSMREEYRKEQQSAVDADDGHFVSIELE (SEQ ID NO: 5)
M2-4: M2del FG#1; delete M2’s 44-54 aa (delete nucleotides 843-875; 11 aa)
MSLLTEVETPIRNEWGCRCNGSSDPLTIAANIIGILHLTLWILFKYGLKGGPSTEGVPKS
MREEYRKEQQSAVDADDGHFVSIELE (SEQ ID NO: 6)
M2-5: M2del FG#2; delete M2’s 44-48 aa (delete nucleotides 843-857; 5 aa)
MSLLTEVETPIRNEWGCRCNGSSDPLTIAANIIGILHLTLWILKCIYRRFKYGLKGGPST
EGVPKSMREEYRKEQQSAVDADDGHFVSIELE (SEQ ID NO: 7)
28
48204255.2
M2-6: M2del FG#3; delete M2’s 44 and 45 aa (delete nucleotides 843-848; 2 aa)
MSLLTEVETPIRNEWGCRCNGSSDPLTIAANIIGILHLTLWILLFFKCIYRRFKYGLKGG
PSTEGVPKSMREEYRKEQQSAVDADDGHFVSIELE (SEQ ID NO: 8)
[0123] M2-4 (M2del FG#1) was generated but was not passagable in normal MDCK cells, but
may be passagable in a modified host cell (e.g., a cell expressing a wild-type M2 polypeptide).
M2-5 (M2del FG#2) and M2-6 (FG#3) were generated and passaged in normal MDCK cells.
The nucleotide sequence of the M gene of these viruses are stable at least to passage 10 in
MDCK cells. These mutants could be propagated and passaged in other cells as well (e.g., cells
that support influenza replication). It was also found that these mutants are not attenuated and
are pathogenic.
[0124] As described in the Examples below, the M2 mutant viruses described herein do not
replicate in the respiratory tract or disseminate to other organs in the ferret model and are not
transmitted in the ferret model. Vaccines comprising M2 mutant elicit robust immune responses
in mammals and protect mammals against influenza virus challenge. M2KO virus elicits both
humoral and mucosal immune responses in mice, and protects mice from lethal homosubtypic
and heterosubtypic challenge. Vaccines comprising M2 mutant virus as described herein provide
effective protection against influenza challenge and have the advantage of being attenuated in
mammalian hosts. These findings demonstrate that the M2 mutant viruses described herein are
useful for vaccines against influenza.
IV. Cell-based virus production system
A. Producing “first generation” mutant viruses
[0125] Mutant virus, such as those carrying mutant M2 nucleic acid, can be generated by
plasmid-based reverse genetics as described by Neumann et al., Generation of influenza A
viruses entirely from clone cDNAs, Proc. Natl. Acad. Sci. USA 96:9345-9350 (1999), herein
29
48204255.2
incorporated by reference in its entirety. Briefly, eukaryotic host cells are transfected with one
or more plasmids encoding the eight viral RNAs. Each viral RNA sequence is flanked by an
RNA polymerase I promoter and an RNA polymerase I terminator. Notably, the viral RNA
encoding the M2 protein includes the mutant M2 nucleic acid sequence. The host cell is
additionally transfected with one or more expression plasmids encoding the viral proteins (e.g.,
polymerases, nucleoproteins and structural proteins), including a wild-type M2 protein.
Transfection of the host cell with the viral RNA plasmids results in the synthesis of all eight
influenza viral RNAs, one of which harbors the mutant M2 sequence. The co-transfected viral
polymerases and nucleoproteins assemble the viral RNAs into functional vRNPs that are
replicated and transcribed, ultimately forming infectious influenza virus having a mutant M2
nucleic acid sequence, yet having a functional M2 polypeptide incorporated into the viral lipid
envelope.
[0126] Alternative methods of producing a “first generation” mutant virus include a
ribonucleoprotein (RNP) transfection system that allows the replacement of influenza virus
genes with in vitro generated recombinant RNA molecules, as described by Enami and Palese,
High-efficiency formation of influenza virus transfectants, J. Virol. 65(5):2711-2713, which is
incorporated herein by reference.
[0127] The viral RNA is synthesized in vitro and the RNA transcripts are coated with viral
nucleoprotein (NP) and polymerase proteins that act as biologically active RNPs in the
transfected cell as demonstrated by Luytjes et al., Amplification, expression, and packaging of a
foreign gene by influenza virus, Cell 59:1107-1113, which is incorporated herein by reference.
[0128] The RNP transfection method can be divided into four steps: 1) Preparation of RNA:
plasmid DNA coding for an influenza virus segment is transcribed into negative-sense RNA in
an in vitro transcription reaction; 2) Encapsidation of the RNA: the transcribed RNA is then
mixed with gradient purified NP and polymerase proteins isolated from disrupted influenza virus
to form a biologically active RNP complex; 3) Transfection and rescue of the encapsidated RNA:
48204255.2
the artificial ribonucleocapsid is transfected to the cells previously infected with a helper
influenza virus that contains a different gene from the one being rescued; the helper virus will
amplify the transfected RNA; 4) Selection of transfected gene: because both the helper virus and
the transfectant containing the rescued gene are in the culture supernatant, an appropriate
selection system using antibodies is necessary to isolate the virus bearing the transfected gene.
[0129] The selection system allows for the generation of novel transfectant influenza viruses
with specific biological and molecular characteristics. Antibody selection against a target
surface protein can then be used for positive or negative selection.
[0130] For example, a transfectant or mutant virus that contains an M2 gene that does not
express an M2 protein can be grown in a suitable mammalian cell line that has been modified to
stably express the wild-type functional M2 protein. To prevent or inhibit replication of the
helper virus expressing the wild-type M2 gene, and therefore the M2e protein at the membrane
surface, antibodies against M2e can be used. Such antibodies are commercially available and
would inhibit the replication of the helper virus and allow for the transfectant/mutant virus
containing the mutant M2 to grow and be enriched in the supernatant. Inhibition of influenza
virus replication by M2e antibodies has been described previously in Influenza A virus M2
protein: monoclonal antibody restriction of virus growth and detection of M2 in virions, J Virol
62:2762-2772 (1988) and Treanor et al, Passively transferred monoclonal antibody to the M2
protein inhibits influenza A virus replication in mice, J. Virol. 64:1375-1377 (1990).
[0131] Additionally or alternatively, the same antibodies can be used to ‘capture’ the helper
virus and allow for the enrichment of the transfectant. For example, the antibodies can be used
to coat the bottom of a tissue culture dish or can be used in a column matrix to allow for
enrichment for the transfectant in the supernatant or eluate.
[0132] The transfectant virus can be grown in M2 expressing cells in multi-well plates by limit
dilution and then be identified and cloned, for example, by creating replica plates. For example,
31
48204255.2
one-half of an aliquot of a given well of the multi-well plate containing the grown virus can be
used to infect MDCK cells and the other half to infect MDCK cells that express M2 protein.
Both the transfectant virus and helper virus will grow in MDCK cells that express M2 protein.
However, only helper virus will grow in standard MDCK cells allowing for identifying the well
in the multi-well plate that contains the transfectant. The transfectant virus can be further plaque
purified in the cells that express M2 protein.
B. Propagating viral mutants
[0133] In some embodiments, viral mutants described herein are maintained and passaged in
host cells. By way of example, but not by way of limitation, exemplary host cells appropriate for
growth of influenza viral mutants, such as influenza A viral mutants include any number of
eukaryotic cells, including, but not limited to Madin-Darby canine kidney cells (MDCK cells),
simian cells such as African green monkey cells (e.g., Vero cells), CV-1 cells and rhesus monkey
kidney cells (e.g., LLcomk.2 cells), bovine cells (e.g., MDBK cells), swine cells, ferret cells
(e.g., mink lung cells) BK-1 cells, rodent cells (e.g., Chinese Hamster Ovary cells), human cells,
e.g., embryonic human retinal cells (e.g., PER-C6®), 293T human embryonic kidney cells and
avian cells including embryonic fibroblasts.
[0134] Additionally or alternatively, in some embodiments, the eukaryotic host cell is modified
to enhance viral production, e.g., by enhancing viral infection of the host cell and/or by
enhancing viral growth rate. For example, in some embodiments, the host cell is modified to
express, or to have increased expression, of 2,6-linked sialic acid on the cell surface, allowing for
more efficient and effective infection of these cells by mutant or wild-type influenza A viruses.
See e.g., U.S. Patent Publication No. 2010-0021499, and U.S. Patent No. 7,176,021, herein
incorporated by reference in their entirety. Thus, in some illustrative embodiments, Chinese
Hamster Ovary Cells (CHO cells) and/or Vero cells modified to express at least one copy of a
2,6-sialyltransferase gene (ST6GAL 1) are used. By way of example, but not by way of
limitation, the Homo sapiens ST6 beta-galatosamide alpha-2,6-sialyltransferase gene sequence
32
48204255.2
denoted by the accession number BC040009.1, is one example of a ST6Gal gene that can be
integrated into and expressed by a CHO cell. One or more copies of a polynucleotide that
encodes a functional ST6Gal I gene product can be engineered into a cell. That is, cells which
have been stably transformed to express 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 copies
of a ST6Gal I gene may be used. A single expression cassette may include one or more copies of
the ST6Gal I gene to be expressed, which is operably linked to regulatory elements, such as
promoters, enhancers, and terminator and polyadenylation signal sequences, to facilitate the
expression of the ST6Gal I gene or its copies. Alternatively, a single expression cassette may be
engineered to express one copy of an ST6Gal I gene, and multiple expression cassettes integrated
into a host cell genome. Accordingly, in some embodiments, at least one ST6Gal I gene is
incorporated into the genome of a host cell, such that the cell expresses the ST6Gal I gene and its
enzymatic protein product. Depending on the copy number, a single host cell may express many
functional ST6Gal I gene proteins.
[0135] Suitable vectors for cloning, transfecting and producing stable, modified cell lines are
well known in the art. One non-limiting example includes the pcDNA3.1 vectors (Invitrogen).
[0136] Additionally or alternatively, in some embodiments, the eukaryotic host cell is modified
to produce a wild-type version of a mutant viral gene, thereby providing the gene to the virus in
trans. For example, a viral strain harboring a mutant M2 protein may exhibit an enhanced
growth rate (e.g., greater viral production) when passaged in host cells producing the wild-type
M2 protein. In some embodiments, the a viral strain harboring a mutant M2 protein may not
grow or replicate in a cell which does not express a wild-type M2 gene. In addition, such host
cells may slow or prevent viral reversion to a functional M2 sequence, because, for example,
there is no selective pressure for reversion in such a host.
[0137] Method for producing both expression vectors and modified host cells are well known
in the art. For example, an M2 expression vector can be made by positioning the M2 nucleic
33
48204255.2
acid sequence (M2 ORF sequence; this is “wild-type” M2’s start codon to stop codon (Table 5))
below in a eukaryotic expression vector.
Table 5: Wild-type M2 nucleic acid sequence
atgagtcttctaaccgaggtcgaaacgcctatcagaaacgaatgggggtgcagatgcaacggttcaagtgatcctctcactattgccgcaaa
tatcattgggatcttgcacttgacattgtggattcttgatcgtctttttttcaaatgcatttaccgtcgctttaaatacggactgaaaggagggccttc
tacggaaggagtgccaaagtctatgagggaagaatatcgaaaggaacagcagagtgctgtggatgctgacgatggtcattttgtcagcata
gagctggagtaa (SEQ ID NO: 9)
[0138] Host cells (e.g., MDCK cells) can then be transfected by methods known in the art, e.g.,
using commercially available reagents and kits, such as TransIT® LT1 (Mirus Bio, Madison,
WI). By way of example, but not by way of limitation, cells can be selected and tested for M2
expression by cotransfection with a detectable marker or a selectable marker (e.g., hygromycinresistance) and/or by screening, for example, with indirect immunostaining using an M2
antibody. M2 expression can be determined by indirect immunostaining, flow cytometry or
ELISA.
[0139] By way of example, but not by way of limitation, 293T human embryonic kidney cells
and Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum and in minimal essential medium (MEM)
containing 5% newborn calf serum, respectively. All cells were maintained at 37ºC in 5% CO2.
Hygromycin-resistant MDCK cells stably expressing M2 protein from A/Puerto Rico/8/34
(H1N1) were established by cotransfection with plasmid pRHyg, containing the hygromycin
resistance gene, and plasmid pCAGGS/M2, expressing the full-length M2 protein, at a ratio of
1:1. The stable MDCK cell clone (M2CK) expressing M2 was selected in medium containing
0.15 mg/mL of hygromycin (Roche, Mannheim, Germany) by screening with indirect
immunostaining using an anti-M2 (14C2) monoclonal antibody (Iwatsuki et al., JVI, 2006,
vol.80, No.1, p.5233-5240). The M2CK cells were cultured in MEM supplemented with 10%
34
48204255.2
fetal calf serum and 0.15 mg/mL of hygromycin. In M2CK cells, the expression levels and
localization of M2 were similar to those in virus-infected cells (data not shown). M2 expressing
Vero cells can be made in a similar fashion.
[0140] In some embodiments, cells and viral mutants are cultured and propagated by methods
well known in the art. By way of example, but not by way of limitation, in some embodiments,
host cells are grown in the presence of MEM supplemented with 10% fetal calf serum. Cells
expressing M2 are infected at an MOI of 0.001 by washing with PBS followed by adsorbing
virus at 37ºC. In some embodiments, viral growth media containing trypsin/TPCK is added and
the cells are incubated for 2-3 days until cytopathic effect is observed.
[0141] Along these lines, disposable bioreactor systems have been developed for mammalian
cells, with or without virus, whose benefits include faster facility setup and reduced risk of crosscontamination. The cells described herein, for instance, can be cultured in disposable bags such
as those from Stedim, Bioeaze bags from SAFC Biosciences, HybridBagTM from Cellexus
Biosytems, or single use bioreactors from HyClone or Celltainer from Lonza. Bioreactors can
be 1 L, 10 L, 50 L, 250 L, 1000 L size formats. In some embodiments, the cells are maintained
in suspension in optimized serum free medium, free of animal products. The system can be a
fed-batch system where a culture can be expanded in a single bag from 1 L to 10 L for example,
or a perfusion system that allows for the constant supply of nutrients while simultaneously
avoiding the accumulation of potentially toxic by-products in the culture medium.
[0142] For long term storage, mutant virus can be stored as frozen stocks.
V. Vaccines and method of administration
A. Immunogenic compositions / vaccines
[0143] There are various different types of vaccines which can be made from the cell-based
virus production system disclosed herein. The present disclosure includes, but is not limited to,
48204255.2
the manufacture and production of live attenuated virus vaccines, inactivated virus vaccines,
whole virus vaccines, split virus vaccines, virosomal virus vaccines, viral surface antigen
vaccines and combinations thereof. Thus, there are numerous vaccines capable of producing a
protective immune response specific for different influenza viruses where appropriate
formulations of any of these vaccine types are capable of producing an immune response, e.g., a
systemic immune response. Live attenuated virus vaccines have the advantage of being also able
to stimulate local mucosal immunity in the respiratory tract.
[0144] In some embodiments, vaccine antigens used in the compositions described herein are
“direct” antigens, i.e. they are not administered as DNA, but are the antigens themselves. Such
vaccines may include a whole virus or only part of the virus, such as, but not limited to viral
polysaccharides, whether they are alone or conjugated to carrier elements, such as carrier
proteins, live attenuated whole microorganisms, inactivated microorganisms, recombinant
peptides and proteins, glycoproteins, glycolipids, lipopeptides, synthetic peptides, or ruptured
microorganisms in the case of vaccines referred to as “split” vaccines.
[0145] In some embodiments a complete virion vaccine is provided. A complete virion vaccine
can be concentrated by ultrafiltration and then purified by zonal centrifugation or by
chromatography. Typically, the virion is inactivated before or after purification using formalin or
beta-propiolactone, for instance.
[0146] In some embodiments, a subunit vaccine is provided, which comprises purified
glycoproteins. Such a vaccine may be prepared as follows: using viral suspensions fragmented
by treatment with detergent, the surface antigens are purified, by ultracentrifugation for example.
The subunit vaccines thus contain mainly HA protein, and also NA. The detergent used may be
cationic detergent for example, such as hexadecyl trimethyl ammonium bromide, an anionic
detergent such as ammonium deoxycholate; or a nonionic detergent such as that commercialized
under the name TRITON X100. The hemagglutinin may also be isolated after treatment of the
virions with a protease such as bromelin, then purified by standard methods.
36
48204255.2
[0147] In some embodiments, a split vaccine is provided, which comprises virions which have
been subjected to treatment with agents that dissolve lipids. A split vaccine can be prepared as
follows: an aqueous suspension of the purified virus obtained as above, inactivated or not, is
treated, under stirring, by lipid solvents such as ethyl ether or chloroform, associated with
detergents. The dissolution of the viral envelope lipids results in fragmentation of the viral
particles. The aqueous phase is recuperated containing the split vaccine, constituted mainly of
hemagglutinin and neuraminidase with their original lipid environment removed, and the core or
its degradation products. Then the residual infectious particles are inactivated if this has not
already been done.
[0148] In some embodiments, inactivated influenza virus vaccines are provided. In some
embodiments, the inactivated vaccines are made by inactivating the virus using known methods,
such as, but not limited to, formalin or ß-propiolactone treatment. Inactivated vaccine types that
can be used as described herein can include whole-virus (WV) vaccines or subvirion (SV) (split)
vaccines. The WV vaccine contains intact, inactivated virus, while the SV vaccine contains
purified virus disrupted with detergents that solubilize the lipid-containing viral envelope,
followed by chemical inactivation of residual virus.
[0149] Additionally or alternatively, in some embodiments, live attenuated influenza virus
vaccines are provided. Such vaccines can be used for preventing or treating influenza virus
infection, according to known method steps.
[0150] In some embodiments, attenuation is achieved in a single step by transfer of attenuated
genes from an attenuated donor virus to an isolate or reassorted virus according to known
methods (see, e.g., Murphy, Infect. Dis. Clin. Pract. 2, 174 (1993)). In some embodiments, a
virus is attenuated by mutation of one or more viral nucleic acid sequences, resulting in a mutant
virus. For example, in some embodiments, the mutant viral nucleic acid sequence codes for a
defective protein product. In some embodiments, the protein product has diminished function or
37
48204255.2
no function. In other embodiments, no protein product is produced from the mutant viral nucleic
acid.
[0151] The virus can thus be attenuated or inactivated, formulated and administered, according
to known methods, as an immunogenic composition (e.g., as a vaccine) to induce an immune
response in an animal, e.g., an avian and/or a mammal. Methods are well-known in the art for
determining whether such attenuated or inactivated vaccines have maintained similar antigenicity
to that of the clinical isolate or a high growth strain derived therefrom. Such known methods
include the use of antisera or antibodies to eliminate viruses expressing antigenic determinants of
the donor virus; chemical selection (e.g., amantadine or rimantidine); HA and NA activity and
inhibition; and DNA screening (such as probe hybridization or PCR) to confirm that donor genes
encoding the antigenic determinants (e.g., HA or NA genes) or other mutant sequences (e.g.,
M2) are not present in the attenuated viruses. See, e.g., Robertson et al., Giornale di Igiene e
Medicina Preventiva, 29, 4 (1988); Kilbourne, Bull. M2 World Health Org., 41, 643 (1969); and
Robertson et al., Biologicals, 20, 213 (1992).
[0152] In some embodiments, the vaccine includes an attenuated influenza virus that lacks
expression of a functional M2 protein. In some embodiments, the mutant virus replicates well in
cells expressing M2 proteins, but in the corresponding wild-type cells, expresses viral proteins
without generating infectious progeny virions.
[0153] Pharmaceutical compositions of the present invention, suitable for intradermal
administration, inoculation or for parenteral or oral administration, comprise attenuated or
inactivated influenza viruses, and may optionally further comprising sterile aqueous or nonaqueous solutions, suspensions, and emulsions. The compositions can further comprise auxiliary
agents or excipients, as known in the art. See, e.g., Berkow et al., The Merck Manual, 15th
edition, Merck and Co., Rahway, N.J. (1987); Goodman et al., eds., Goodman and Gilman's The
Pharmacological Basis of Therapeutics, Eighth Edition, Pergamon Press, Inc., Elmsford, N.Y.
(1990); Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and
38
48204255.2
Therapeutics, Third Edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. (1987);
and Katzung, ed., Basic and Clinical Pharmacology, Fifth Edition, Appleton and Lange,
Norwalk, Conn. (1992).
[0154] In some embodiments, preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and/or emulsions, which may contain auxiliary
agents or excipients known in the art. Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl
oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance
antigen absorption. Liquid dosage forms for oral administration may generally comprise a
liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes
include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly
used in the art, such as purified water. Besides the inert diluents, such compositions can also
include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring,
or perfuming agents.
[0155] When a composition of the present invention is used for administration to an individual,
it can further comprise salts, buffers, adjuvants, or other substances which are desirable for
improving the efficacy of the composition. For vaccines, adjuvants, substances that augment a
specific immune response, can be used. Normally, the adjuvant and the composition are mixed
prior to presentation to the immune system, or presented separately, but into the same site of the
organism being immunized.
[0156] In some embodiments, the immunogenic compositions (e.g., vaccines) disclosed herein
include multiple, different types of virus or viral antigens, at least one of which includes a mutant
M2 gene (e.g., a virus comprising the M2KO(ΔTM) (SEQ ID NO:1) mutation), and/or a
corresponding mutation in the M2 functional equivalent of that virus (e.g., the NB protein of
influenza B, or the CM1 protein of influenza C). In other embodiments, the immunogenic
compositions include a single type of virus or viral antigen which includes a mutant M2 gene
39
48204255.2
(e.g., a virus comprising the M2KO(ΔTM) (SEQ ID NO:1) mutation) and/or a corresponding
mutation in the M2 functional equivalent of that virus (e.g., the NB protein of influenza B, or the
CM1 protein of influenza C). For example, in some embodiments, the main constituent of an
immunogenic compositions such as a vaccine composition includes one or more influenza
viruses of type A, B or C, or any combination thereof or any combination of antigens from these
viruses, wherein at least one virus includes a mutant M2 gene (e.g., a virus comprising the
M2KO(ΔTM) (SEQ ID NO:1) mutation) and/or a corresponding mutation in the M2 functional
equivalent of that virus (e.g., the NB protein of influenza B, or the CM1 protein of influenza
C)For example, in some embodiments, at least two of the three types, at least two of different
subtypes, at least two of the same type, at least two of the same subtype, or a different isolate(s)
or reassortant(s) are provided in an immunogenic composition (e.g., a vaccine). By way of
example, but not by way of limitation, human influenza virus type A includes H1N1, H2N2 and
H3N2 subtypes. In some embodiments, the immunogenic compositions (e.g., vaccines) include
a virus comprising a mutant M2 gene (e.g., a virus comprising the M2KO(ΔTM) (SEQ ID NO:1)
mutation) and/or a corresponding mutation in the M2 functional equivalent of that virus (e.g., the
NB protein of influenza B, or the CM1 protein of influenza C) and about 0.1 to 200 µg, e.g., 10
to 15 µg of hemagglutinin from each of the strains entering into the composition. Heterogeneity
in a vaccine may be provided by mixing replicated influenza viruses for at least two influenza
virus strains, such as from 2-50 strains, or any range or value therein. In some embodiments,
influenza A or B virus strains having a modern antigenic composition are used. In addition,
immunogenic compositions (e.g., vaccines) can be provided for variations in a single strain of an
influenza virus, using techniques known in the art.
[0157] In some embodiments, the vaccine comprises a virus comprising the M2KO(ΔTM)
(SEQ ID NO:1) mutation together with other viral components and/or genes expressing other
viral components. In some embodiments, the vaccine (e.g., a virus comprising the M2KO(ΔTM)
(SEQ ID NO:1) mutation) comprises genes from other viral strains, including but not limited to,
for example, HA and NA genes from other viral strains. In some embodiments, the vaccine
40
48204255.2
comprises HA and NA genes from human influenza virus type A subtypes H5N1, H1N1, H2N2
or H3N2. In some embodiments, the vaccine comprises HA and NA genes from, for example,
PR8xBrisbane/10/2007, A/Vietnam/1203/2004, or A/California/07/2009 (CA07) viruses.
[0158] A pharmaceutical composition according to the present invention may further or
additionally comprise at least one chemotherapeutic compound, e.g., for gene therapy, an
immunosuppressant, an anti-inflammatory agent or an immunostimulatory agent, or anti-viral
agents including, but not limited to, gamma globulin, amantadine, guanidine,
hydroxybenzimidazole, interferon-α, interferon-β, interferon-γ, tumor necrosis factor-α,
thiosemicarbarzones, methisazone, rifampin, ribavirin, a pyrimidine analog, a purine analog,
foscarnet, phosphonoacetic acid, acyclovir, dideoxynucleosides, a protease inhibitor, or
ganciclovir.
[0159] The composition can also contain variable but small quantities of endotoxin-free
formaldehyde, and preservatives, which have been found safe and not contributing to undesirable
effects in the organism to which the composition of the invention is administered.
B. Administration
[0160] An immunogenic composition (e.g., vaccine) as disclosed herein may be administered
via any of the routes conventionally used or recommended for vaccines: parenteral route,
mucosal route, and may be in various forms: injectable or sprayable liquid, formulation which
has been freeze-dried or dried by atomization or air-dried, etc. Vaccines may be administered by
means of a syringe or by means of a needle-free injector for intramuscular, subcutaneous or
intradermal injection. Vaccines may also be administered by means of a nebulizer capable of
delivering a dry powder or a liquid spray to the mucous membranes, whether they are nasal,
pulmonary, vaginal or rectal.
[0161] A vaccine as disclosed herein may confer resistance to one or more influenza strains by
either passive immunization or active immunization. In active immunization, an inactivated or
41
48204255.2
attenuated live vaccine composition is administered prophylactically to a host (e.g., a mammal),
and the host's immune response to the administration protects against infection and/or disease.
For passive immunization, the elicited antisera can be recovered and administered to a recipient
suspected of having an infection caused by at least one influenza virus strain.
[0162] Also described are methods for preventing or attenuating a disease or disorder, e.g.,
infection by at least one influenza virus strain. As used herein, a vaccine is said to prevent or
attenuate a disease if its administration results either in the total or partial attenuation (i.e.,
suppression) of a symptom or condition of the disease, or in the total or partial immunity of the
individual to the disease.
[0163] At least one inactivated or attenuated influenza virus, or composition thereof, as
described may be administered by any means that achieve the intended purposes, using a
pharmaceutical composition as previously described. For example, administration of such a
composition may be by various parenteral routes such as subcutaneous, intravenous, intradermal,
intramuscular, intraperitoneal, intranasal, oral or transdermal routes. Parenteral administration
can be by bolus injection or by gradual perfusion over time. In some embodiments, an
immunogenic composition as disclosed herein is by intramuscular or subcutaneous application.
[0164] In some embodiments, a regimen for preventing, suppressing, or treating an influenza
virus related pathology comprises administration of an effective amount of a vaccine
composition as described herein, administered as a single treatment, or repeated as enhancing or
booster dosages, over a period up to and including between one week and about 24 months, or
any range or value therein. In some embodiments, an influenza vaccine as disclosed herein is
administered annually.
[0165] According to the present disclosure, an "effective amount" of a vaccine composition is
one that is sufficient to achieve a desired biological effect. It is understood that, in some
embodiments, the effective dosage will be dependent upon the age, sex, health, and weight of the
42
48204255.2
recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the
effect wanted. The ranges of effective doses provided below are not intended to be limiting and
represent exemplary dose ranges. Thus, in some embodiments, the dosage will be tailored to the
individual subject, as is understood and determinable by one of skill in the art. The dosage of an
attenuated virus vaccine for a mammalian (e.g., human) adult can be from about 103-107 plaque
forming units (PFU), or any range or value therein. The dose of inactivated vaccine can range
from about 0.1 to 200, e.g., 50 µg of hemagglutinin protein. However, the dosage should be a
safe and effective amount as determined by conventional methods, using existing vaccines as a
starting point.
C. Intracutaneous delivery
[0166] Live flu vaccines are traditionally delivered intranasally to mimic the natural route of
infection and promote a similar immune response to that of natural virus infection. As an
alternative, disclosed herein are intradermal delivery methods which involve the use of a novel
microneedle device to capitalize on the immunological benefits of intradermal delivery. In some
embodiments, an attenuated virus (e.g., an M2 viral mutant) is used in a vaccine composition for
intradermal administration. In some embodiments, an M2 viral mutant, which does not produce
infectious progeny virus, is provided in a vaccine. Thus, any potential of reassortment with
wild-type circulating influenza viruses is virtually eliminated.
[0167] In embodiments disclosed herein, intradermal delivery (intracutaneous) administers
vaccine to the skin. In some embodiments, intradermal delivery is performed using a
microneedle delivery device. As disclosed herein, intracutaneous delivery has numerous
advantages. For example, the immunogenicity of the vaccine is enhanced by triggering the
immunological potential of the skin immune system. The vaccine has direct access to the potent
antigen-presenting dendritic cells of the skin, i.e., epidermal Langerhans Cells and dermal
dendritic cells. Skin cells produce proinflammatory signals which enhance the immune
response to antigens introduced through the skin. Further, the skin immune system produces
43
48204255.2
antigen-specific antibody and cellular immune responses. Intradermal delivery allows for
vaccine dose sparing, i.e., lower doses of antigen may be effective, in light of the above factors,
when delivered intracutaneously.
[0168] And, because the vaccine is delivered to the skin through the device’s microneedle
array, the risk of unintended needle-sticks is reduced, and intracutaneous vaccine delivery via
microneedle array is relatively painless compared to intramuscular injections with conventional
needle and syringe.
[0169] Microneedle devices are known in the art, are known in the art, including, for example,
those described in published U.S. patent applications 2012/0109066, 2011/0172645,
2011/0172639, 2011/0172638, 2011/0172637, and 2011/0172609. Microneedle devices may be
made, for example, by fabrication from stainless steel sheets (e.g., Trinity Brand Industries,
Georgia; SS 304; 50 µm thick) by wet etching. In some embodiments, individual microneedles
have a length of between about 500µm and 1000µm, e.g., about 750µm, and a width of between
about 100 µm to 500 µm, e.g., about 200 µm. Vaccine can then be applied to the microneedles
as a coating. By way of example, but not by way of limitation, a coating solution may include
1% (w/v) carboxymethyl cellulose sodium salt (low viscosity, USP grad; Carbo-Mer, San Diego
CA), 0.5% (w/v) Lutrol F-68 NF (BASF, Mt. Olive, NJ) and the antigen (e.g., soluble HA
protein at 5 ng/ml; live, attenuated virus such as the M2 mutant virus described herein, etc.). To
reach a higher vaccine concentration, the coating solution may be evaporated for 5 to 10 minutes
at room temperature (~23ºC). Coating may be performed by a dip coating process. The amount
of vaccine per row of microneedles can be determined by submerging the microneedles into 200
µl of phosphate-buffered saline (PBS) for 5 minutes and assaying for the antigen by methods
known in the art.
[0170] In some embodiments, a microneedle device is used that is made mainly of
polypropylene and stainless steel first-cut pieces that fit together with simple snap fits and heat
seals. In some embodiments, the device is completely self-contained and includes the vaccine, a
44
48204255.2
pump mechanism, an activation mechanism, and a microneedle unit. These components are
hidden within a plastic cover. With the device, vaccine infusion is initiated by pressing an
actuation button. Pressing the button simultaneously inserts the microneedles into the skin and
initiates the pumping mechanism that exerts pressure on the primary drug container. When a
spring mechanism exerts sufficient pressure on the vaccine reservoir, vaccine begins to flow
through the microneedle array, and into the skin. In some embodiments, the delivery of the
vaccine dose is completed within about 2 minutes after actuation of the device. After infusion is
complete, the device is gently removed from the skin.
[0171] In some embodiments, a method for intracutaneous administration of an immunogenic
composition (e.g., a vaccine) is provided using a microneedle device. In some embodiments, the
microneedle device comprises a puncture mechanism and an immunogenic composition layer
comprising a plurality of microneedles capable of puncturing skin and allowing an immunogenic
composition to be administered intracutaneously. In some embodiments, the method comprises
depressing the puncture mechanism. In some embodiments, the immunogenic composition (e.g.
vaccine) comprises a virus comprising a nucleic acid sequence encoding a mutant M2 protein
that is expressed or a mutant M2 protein that is not expressed; wherein the expressed mutant M2
protein comprises, or consists of, the amino acid sequence of SEQ ID NO: 4. In some
embodiments, the microneedle array is initially positioned inside of a device housing, and upon
actuation of a lever allows the microneedles to extend through the device bottom and insert into
the skin thereby allowing infusion of the vaccine fluid into the skin.
[0172] The delivery device described herein may be utilized to deliver any substance that may
be desired. In one embodiment, the substance to be delivered is a drug, and the delivery device
is a drug delivery device configured to deliver the drug to a subject. As used herein the term
“drug” is intended to include any substance delivered to a subject for any therapeutic,
preventative or medicinal purpose (e.g., vaccines, pharmaceuticals, nutrients, nutraceuticals,
etc.). In one such embodiment, the drug delivery device is a vaccine delivery device configured
45
48204255.2
to deliver a dose of vaccine to a subject. In one embodiment, the delivery device is configured to
deliver a flu vaccine. The embodiments discussed herein relate primarily to a device configured
to deliver a substance transcutaneously. In some embodiments, the device may be configured to
deliver a substance directly to an organ other than the skin.
EXAMPLES
[0173] While the following examples are demonstrated with influenza A, it is understood that
the mutations and methods described herein are equally applicable to other viruses which express
an M2, an M2-like protein or a protein with the same or similar function as the influenza A M2
protein.
Example 1: Generation of M2 viral mutants
[0174] M2 mutants were constructed as follows.
a) M2-1: M2 ectodomain + 2 stop codons + TM deletion (PR8 M segment +
2 stops (786-791) without 792-842 (TM))
[0175] Partial wild-type M genes from PR8 were amplified by PCR using oligo set 1 and oligo
set 2 as shown below.
TABLE 6
Oligo Set 1
acacacCGTCTCTAGgatcgtctttttttcaaatgcatttacc (SEQ ID NO: 10)
CACACACGTCTCCTATTAGTAGAAACAAGGTAGTTTTT (SEQ ID NO: 11)
Oligo Set 2
acacacCGTCTCatcCTATTAatcacttgaaccgttgc (SEQ ID NO: 12)
46
48204255.2
CACACACGTCTCCGGGAGCAAAAGCAGGTAG (SEQ ID NO: 13)
[0176] The PCR products were then digested with BsmBI. An expression vector (pHH21) was
also digested with BsmBI, and the digested PCR products were ligated into the vector using T4
DNA ligase. E. coli cells were transformed with the vector, and after appropriate incubation,
vectors were isolated and purified by methods known in the art. The mutant M2 portion of the
vector was characterized by nucleic acid sequencing.
b) M2-2: M2 ectodomain + 2 stops + splice defect (PR8 M segment + 2 stops
(786-791) +splice defect nt 51)
[0177] Partial wild-type M genes from PR8 were amplified by PCR using the primer set shown
below.
TABLE 7
PCR primers
’acacacCGTCTCcCTACGTACTCTCTATCATCCCG (SEQ ID NO: 14)
’CACACACGTCTCCTATTAGTAGAAACAAGGTAGTTTTT (SEQ ID NO: 15)
[0178] The PCR products were then digested with BsmBI. An expression vector (pHH21) was
also digested with BsmBI. A double-stranded DNA fragment was then made by annealing the
two nucleotides shown below.
TABLE 8
Annealing nucleotides
’GGGAGCAAAAGCAGGTAGATATTGAAAGatgagtcttctaaccgaggtcgaaac (SEQ ID NO: 16)
47
48204255.2
’GTAGgtttcgacctcggttagaagactcatCTTTCAATATCTACCTGCTTTTGC (SEQ ID NO: 17)
[0179] The digested vector, PCR product and double-stranded fragment were then ligated
using T4 DNA ligase. E. coli cells were transformed with the vector, and after appropriate
incubation, the vectors were isolated and purified by methods known in the art. The mutant M2
portion of the vector was characterized by nucleic acid sequencing.
c) M2-3: M2 ectodomain + 2 stops + splice defect + TM deletion (PR8 M
segment + 2 stops (786-791) without 792-842 (TM)+splice defect nt 51)
[0180] The partial M2-1 mutant (M2 ectodomain + 2 stop codons + TM deletion (PR8 M
segment + 2 stops (786-791) without 792-842 (TM)) was amplified from PR8 by PCR using the
following primers:
TABLE 9
PCR primers
’acacacCGTCTCcCTACGTACTCTCTATCATCCCG (SEQ ID NO: 18)
’CACACACGTCTCCTATTAGTAGAAACAAGGTAGTTTTT (SEQ ID NO: 19)
[0181] The PCR products were then digested with BsmBI. An expression vector (pHH21) was
also digested with BsmBI. A double-stranded DNA fragment was then made by annealing the
two nucleotides shown below.
TABLE 10
Annealing nucleotides
’GGGAGCAAAAGCAGGTAGATATTGAAAGatgagtcttctaaccgaggtcgaaac (SEQ ID NO: 20)
48
48204255.2
’GTAGgtttcgacctcggttagaagactcatCTTTCAATATCTACCTGCTTTTGC (SEQ ID NO: 21)
[0182] The digested vector, PCR product and double-stranded fragment were then ligated
using T4 DNA ligase. E. coli cells were transformed with the vector, and after appropriate
incubation, the vectors were isolated and purified by methods known in the art. The mutant M2
portion of the vector was characterized by nucleic acid sequencing.
[0183] The sequence of each of the three M2 mutant constructs is provided in Tables 1-3.
Example 2: Generation and culturing of M2 mutant virus
[0184] This example demonstrates the culturing of the PR8 virus comprising the M2KO(ΔTM)
(SEQ ID NO:1) mutation. Mutant viruses were generated as reported in Neumann et al.,
Generation of influenza A viruses entirely from clone cDNAs, Proc. Natl. Acad. Sci. USA
96:9345-9350 (1999), with some modifications. Briefly, 293T cells were transfected with 17
plasmids: 8 PolI constructs for 8 RNA segments, one of which harbors the mutant M2 sequence,
and 9 protein-expression constructs for 5 structural proteins as follows: NP (pCAGGS-WSNNP0/14); M2 (pEP24c); PB1 (pcDNA774); PB2 (pcDNA762); and PA (pcDNA787) of A/Puerto
Rico/8/34 (H1N1) virus.
[0185] The plasmids were mixed with transfection reagent (2 µL of Trans IT® LT-1 (Mirus,
Madison, Wis.) per µg of DNA), incubated at room temperature for 15-30 minutes, and added to
1x106 293T cells. Forty-eight hours later, viruses in the supernatant were serially diluted and
inoculated into M2CK cells. Two to four days after inoculation, viruses in supernatant of the last
dilution well in which cells showing clear cytopathic effect (CPE) were inoculated into M2CK
cells for the production of stock virus. The M genes of generated viruses were sequenced to
confirm the gene and the presence of the intended mutations and to ensure that no unwanted
mutations were present.
49
48204255.2
[0186] Mutant M2 viruses were grown and passaged as follows. M2CK host cells were grown
in the presence of MEM supplemented with 10% fetal calf serum. Cells were infected at an MOI
of 0.001 by washing with PBS followed by adsorbing virus at 37ºC. Virus growth media
containing trypsin/TPCK was added and the cells were incubated for 2-3 days until cytopathic
effect was observed.
Example 3 M2KO Replication is Restricted in Normal Cells
[0187] Growth kinetics of the PR8 virus with the M2KO(ΔTM) (SEQ ID NO:1) mutation and
wild-type PR8 were analyzed in both normal MDCK cells and MDCK cells stably expressing
M2 protein (M2CK). Cells were infected with viruses at multiplicity of infection of 10-5. Virus
titers in cell supernatant were determined in MDCK or M2CK cells. Wild-type PR8 grew to
high titers in both cell types whereas M2KO grew well only in M2CK cells and not at all in
MDCK cells (Figure 4).
Example 4: M2KO Virus Produces Viral Antigens, But Not M2, in Normal Cells
[0188] This example demonstrates that the PR8 virus with the M2KO(ΔTM) (SEQ ID NO:1)
mutation produces viral antigens, but not M2 protein, in normal cells. Viral protein expression
was evaluated by infecting wild-type MDCK cells with wild-type PR8 or M2KO at a multiplicity
of infection (MOI) of 0.5 in medium without trypsin to ensure that viruses complete only one life
cycle. Viral proteins in the cell lysates were separated on a 4-12% SDS-PAGE gel and detected
by Western blot using PR8 infected mouse sera (Panel A) or anti-M2 monoclonal antibody
(14C2, Santa Cruz Biotechnology) (Panel B). Figure 3A shows that antisera against PR8 detects
similar levels of protein expression for both PR8 and M2KO. When the lysates are probed with
an anti-M2 monoclonal antibody (Panel B), M2 expression is detected only in PR8 infected cells,
not M2KO. These results indicate that M2KO virus expresses all viral proteins, except M2
protein, to similar levels as PR8 virus (Figure 5)
50
48204255.2
Example 5: M2 mutants Are Attenuated In Vivo
[0189] An experiment was performed to demonstrate that M2 mutant viruses are attenuated in
vivo. Six weeks old BALB/c, female mice (23 per group) were inoculated intranasally with one
of the following mutants: M2KO(yk) as described in J. Virol (2009) 83:5947-5950; M2-1 (TM
del M2KO aka M2KO(ΔTM)) and M2-2 (Splice def M2KO) (collectively termed “M2KO
variants”). The mutant was administered at a dose of 1.2x104 pfu per mouse. A control group of
mice was given PBS. The mice were observed for 14 days after inoculation for any change in
body weight and symptoms of infection. Additionally, after 3 days post-inoculation, virus titers
were taken from the lungs and nasal turbinates (NT) from 3 mice in each group.
[0190] As shown in Figure 6, mice inoculated with the M2KO variants and PBS did not show
any clinical symptoms of infection nor lose any body weight over the 14 day period. The change
in body weight between the groups were comparable over the 14 day period. Additionally, no
virus was detected in the titers that were gathered from the lungs and NT. Together, the lack of
clinical symptoms, lack of loss of body weight and absence of virus indicate that the M2 mutant
viruses are attenuated and not pathogenic in mice.
Example 6: M2 Mutants Induce Antibodies Against Influenza Virus and Protect Mice From
Lethal Virus Challenge
[0191] Testing was also performed to determine antibody titers from the mice described in
Example 5 above and their survival after being challenged with a lethal viral dose. Serum
samples were taken 3 weeks after inoculation and anti-virus IgG antibody titers from the serum
samples were determined by enzyme-linked immunosorbent assay (ELISA). The humoral
response is shown in Figure 7, which shows that all three M2 mutants elevated anti-influenza
virus antibodies higher than the control PBS group.
[0192] In addition, half of the mice within each of the groups were boosted 28 days after
inoculation with same amount of M2 mutant virus. Serum was then collected 6 weeks after the
51
48204255.2
first inoculation and IgG titers against the virus were determined. As shown in Figure 7B, mice
boosted by M2 mutant viruses had a higher level of anti-influenza virus antibodies than ones
were not boosted.
[0193] 49 days after the first inoculation (3 weeks after the boost), the mice were challenged
with a lethal dose of PR8 virus (40 mouse 50% lethal dose (MLD50)). As shown in Figure 8 and
Figure 9, all mice vaccinated with the M2KO variants survived the challenge and lost no weight.
The control mice that were given only PBS, however, lost body weight and did not survive 8
days past the challenge date. On day 3 after the challenge, lungs and NT were obtained and virus
titers determined in MDCK cells by plaque assay. As depicted in Table 11, lung virus titers in
M2KO variants were at least one log lower than titers in naïve control PBS. And almost no
viruses were detected in nasal turbinates in M2KO variants groups but more than 100,000 PFU/g
were detected in the naïve control PBS group, indicating that the M2 mutant vaccines confer
protection and limits the replication of the challenge virus.
TABLE 11: Virus Titer (log10 PFU/g) in Mouse Tissue After Challenge
[0194] In another experiment, six weeks after immunization, the M2KO(ΔTM) groups were
challenged with homosubtypic or heterosubtypic influenza viruses. Mice were challenged with
Aichi (H3N2) virus and scored for survival for 14 days. Results for the heterotypic challenge are
shown in Figure 16.
52
48204255.2
Example 7: Intradermal Vaccine Delivery
[0195] An experiment was performed to show that intradermal vaccine delivery / immunizing
will protect a subject from influenza. BALB/c female, 6-7 weeks old mice ( 5 per group)
(Harland Laboratories) were inoculated either intranasally (IN), intramuscularly (IM) or
intradmermally (ID) with PR8 virus (3.5x107pfu) at a concentration of 1.8x101, 1.8x102, 1.8x103
or 1.8x104 pfu (50 µl) per mouse. Control mice were also given PBS through the three different
routes of administration. Body weight and survival were monitored for 14 days after inoculation.
For the mouse experiments, allergy syringes with intradermal bevel needles were used.
[0196] Most vaccines are administered by intramuscular or subcutaneous injection using
conventional needles and syringes. However, recent studies demonstrate that intradermal
vaccine delivery achieves better immunogenicity than intramuscular or subcutaneous
administration. Intradermal vaccination delivers antigen directly to the enriched skin immune
system and has been shown to be effective for a range of vaccines, including rabies, hepatitis B
and influenza. Intradermal delivery may also provide dose sparing, achieving the same immune
response using less vaccine than required with intramuscular injection. The current state-of-theart for intradermal delivery (using conventional needles and syringes) is the Mantoux technique,
which requires extensive training, is difficult to perform, and often results in misdirected
(subcutaneous) or incomplete administration. The lack of suitable delivery devices has
hampered intradermal vaccination research and product development even though superior
immune responses with this administration route have been documented.
[0197] As shown in Table 12, IN-inoculated mice succumbed to influenza infection at the
higher doses of 1.8x103 and 1.8x104 pfu per mouse, with complete survival only at the lowest
dose of 1.8x101. However, IM- and ID-inoculated mice at all dosages survived. Table 13 shows
the median lethal dose for mice in the IN-inoculated group (MLD50). Figure 10 shows that IMand ID-inoculated mice inoculated with 1.8x104 pfu of the virus displayed no change in body
53
48204255.2
weight, and shows the lack of survival for IN-inoculated mice inoculated with 1.8x104 pfu of
virus.
Table 12. Mice survival after PR8 inoculation
Virus Dose
(pfu)
Route of Administration
IM ID IN
1.8x101 5/5 5/5 5/5
1.8x102 5/5 5/5 1/5
1.8x103 5/5 5/5 0/5
1.8x104 5/5 5/5 0/5
PBS 5/5 5/5 5/5
Table 13. Median lethal dose for mice (MLD50).
Route MLD50 (pfua/mouse)
IN 76
IM > 1.8x104
ID > 1.8x104
a
pfu: plaque forming unit.
[0198] Serum was collected at 2 weeks (Figure 11A) and 7 weeks (Figure 11B) after
inoculation and evaluated for anti-PR8 IgG antibody as determined by an ELISA. “Hi”
54
48204255.2
represents 1.8x104 pfu inoculations, and “Lo” represents 1.8x101 pfu. The responses of the IN-,
IM- and ID-inoculated mice at both time periods are similar. At each time period, IN-inoculated
mice presented the highest number of antibodies. Only IN-inoculated mice inoculated with
1.8x101 pfu were identified (i.e., “Lo”), because by this time, the IN-inoculated mice inoculated
with higher doses had expired. IM- and ID-inoculated mice presented lower levels of antibodies
than the IN-inoculated mice, although mice inoculated at the higher doses exhibited greater
amounts of antibodies when compared with the control mice given only PBS. Additionally, over
time, the intradermal administration route produced more antibodies than the intramuscular
route, as demonstrated by the higher titer levels shown in Figure 11B.
[0199] In another experiment, groups of IN-, IM- and ID-inoculated mice (5 mice per group,
except for 4 mice in 1.8x103 group) were challenged 8 week after vaccination. Specifically,
1.8x101 IN-inoculated mice, 1.8x103 IM-inoculated mice, 1.8x104 IM-inoculated mice, 1.8x103
ID-inoculated mice and 1.8x104 ID-inoculated mice were challenged. Mice that lost more than
% of their body weight were euthanized.
[0200] As shown in Figure 12, 100% of IM-inoculated mice at a dose of 1.8x103 did not
survive 8 days after the challenge. The survival rate of all ID-inoculated mice was between 40%
and 60%. The survival rate of IM-inoculated mice at 1.8x104, however, was 100%. Figure 13
shows that the ID-inoculated and IM-inoculated (1.8x104) groups of mice had an initial average
weight loss, but ended up with a relative low weight loss from the challenge date.
[0201] An evaluation of the ID-incoulated mice (1.8x104) showed that two mice (1 and 5 in
Figure 14 and Figure 15), elicited a better immune response than the other mice, and further did
not develop symptoms of influenza infection (e.g., body weight loss, rough fur, quietness, etc.).
However, all mice in the IM-inoculated group (1.8x104) showed some symptoms and lost at least
% in body weight.
55
48204255.2
Example 8: Stability of M2KO Variants
[0202] To test the stability of M2 gene of M2KO variants in wild-type cells, the M2KO
variants were passaged in wild-type MDCK cells, which lacks M2 protein expression, along with
M2CK cells which are M2 protein expressing MDCK cells. All M2KO variants were
passageable in M2CK cells without any mutations until at least passage 10. Although, M2-1
(TM del M2KO), M2-2 (Splice def M2KO), and M2-3 (TM del + Splice def M2KO) were not
able to be passaged in wild-type MDCK cells (no cytopathic effect (CPE) is seen in wild-type
MDCK cells), M2KO(yk) showed CPE even after 4th passage in MDCK cells. M segment
RNAs were extracted from M2KO(yk) passage 4 in wild-type MDCK and the cDNA were
sequenced. As shown in Table 14, two inserted stop codons of M2KO(yk) were edited and
M2KO(yk) passage 4 in wild-type MDCK possessed full length M2 protein gene.
Table 14. Sequence around inserted 2 stop codons (nt 700-800 of M segment, stop codons at
nt 786-791.)
Virus Sequence
Original M2KO(yk) 3’CAACGGTTCAAGTGATTAATAAACTATTGCC
(SEQ ID NO: 22)
M2KO(yk) passage 2 in M2CK 3’CAACGGTTCAAGTGATTAATAAACTATTGCC
(SEQ ID NO: 22)
M2KO(yk) passage 4 in MDCK 3’CAACGGTTCAAGTGATTGGTGGACTGTTGCC
(SEQ ID NO: 23)
Example 9: M2KO Vaccinations
[0203] To demonstrate that the M2KO vaccine can stimulate an immune response similar to a
natural influenza infection, a vaccine experiment was conducted. Natural influenza infection
was represented by a low inoculum of PR8 virus and the standard inactivated flu vaccine was
56
48204255.2
represented by inactivated PR8 virus (Charles River) delivered the standard intramuscular route
and intranasally.
[0204] Six to seven week old BALB/c mice were immunized intranasally with live virus (10
pfu PR8), PR8 virus comprising M2KO(ΔTM) (104 pfu), or 1 µg inactivated PR8 virus, delivered
both intranasally and intramuscularly. Mice given 104 infectious particles of M2KO(ΔTM)
intranasally lost no weight and showed no signs of infection. Furthermore, the lungs of mice
treated with M2KO(ΔTM) contained no detectable infectious particles three days postinoculation. Sera was obtained from the immunized mice on day 21 and antibody titers against
the hemagglutinin were determined by a standard ELISA assay. Figure 17 shows that anti-HA
IgG titers were highest in the live virus and M2KO(ΔTM) groups relative to the inactivated
vaccine groups. Mucosal IgA antibody against influenza was detected in sera only in the live
PR8 or M2KO vaccinated mice.
[0205] Six weeks after immunization, all groups were challenged with homosubtypic (PR8,
H1N1) or heterosubtypic (Aichi, H3N2) influenza viruses. Both M2KO and inactivated
vaccinations protected mice from homosubtypic virus infection (Figure 18). However, only
M2KO vaccinated mice were protected from heterosubtypic virus challenge (Figure 19). The
mice immunized with inactivated vaccine succumbed to infection similar to naïve mice.
Example 10 The M2KO(ΔTM) Virus Does not Replicate In The Respiratory Tract Or Other
Organs
[0206] Summary – This example demonstrates that the M2KO(ΔTM) virus does not replicate
in the respiratory tract or disseminate to other organs in the ferret model. The M2KO(ΔTM)
virus was administered intranasally to 3 male ferrets at a dose level of 1x107 TCID50. As a
control, second group of 3 male ferrets was administered A/Brisbane/10/2007 (H3N2) influenza
A virus intranasally at a dose of 1x107 TCID50. Following virus inoculation, ferrets were
observed until Day 3 post inoculation for mortality, with body weights, body temperatures and
57
48204255.2
clinical signs measured daily. Necropsy was performed on all animals 3 days post inoculation.
Organs were collected for histopathology and viral titers.
[0207] The control group receiving A/Brisbane/10/2007 (H3N2) exhibited a transient reduction
in weight and an increase in body temperature 2 days after inoculation which was not observed
in the M2KO(ΔTM) group. Activity levels were also reduced in the A/Brisbane/10/2007 group
with sneezing observed on days 2-3 post infection. No changes in activity level or clinical signs
associated with virus exposure were observed in the M2KO(ΔTM) group. Histopathological
analysis revealed changes in the nasal turbinates in animals exposed to influenza
A/Brisbane/10/2007 (H3N2) that were not seen in ferrets exposed to the M2KO(ΔTM) virus.
Exposure to A/Brisbane/10/2007 resulted in atrophy of respiratory epithelium, infiltrates of
neutrophils and edema in the nasal turbinates. No other organ was affected by the virus
inoculation. Under the conditions of the experiment, the M2KO(ΔTM) virus did not induce
clinical signs of infection or result in histological changes in the organs analyzed.
MATERIALS AND METHODS
[0208] A. Vaccine Material and Control Virus: The M2KO(ΔTM) virus is a recombinant virus
which possesses internal 6 genes of PR8 (nucleoprotein (NP), polymerase genes (PA, PB1, PB2),
non-structural (NS), matrix (M)), but which does not express functional M2 protein, as well as
HA and NA genes of Influenza A/Brisbane/10/2007-like A/Uruguay/716/2007(H3N2). The
A/Brisbane/10/2007 (H3N2) wild type virus served as the control virus and was supplied by
IITRI. The viruses were kept frozen at -65°C until used.
[0209] B. Test Article and Positive Control Dose Formulation: The M2KO(ΔTM) virus dosing
solution of 1x107 TCID50/mL per 316 µL was prepared by diluting 8 µL of 1x1010 TCID50/mL
into 2.528 mL PBS. The A/Brisbane/10/2007 (H3N2) at a titer of 1x107 TCID50/mL per 316 µL
was used undiluted.
58
48204255.2
[0210] C. Animals and Animal Care: Eight male ferrets were purchased from Triple F Farms
and six of the ferrets were placed on study. Animals were approximately 4 months of age at the
time of study initiation. The animals were certified by the supplier to be healthy and free of
antibodies to infectious diseases. Upon arrival the animals were single housed in suspended wire
cages with slat bottoms, suspended over paper-lined waste pans. The animal room and cages had
been cleaned and sanitized prior to animal receipt, in accordance with accepted animal care
practices and relevant standard operating procedures. Certified Teklad Global Ferret Diet #2072
(Teklad Diets, Madison WI) and city of Chicago tap water were provided ad libitum and were
refreshed at least once daily. Fluorescent lighting in the animal rooms was maintained on a 12-
hr light/dark cycle. Animal room temperature and relative humidity were within respective
protocol limits and ranged from 22.0 to 25.0°C and 33 to 56%, respectively, during the study.
[0211] D. Animal Quarantine and Randomization: The ferrets were held in quarantine for five
days prior to randomization and observed daily. Based on daily observations indicating general
good health of the animals the ferrets were released from quarantine for randomization and
testing. Following quarantine, ferrets were weighed and assigned to treatment groups using a
computerized randomization procedure based on body weights that produced similar group mean
values [ToxData® version 2.1.E.11 (PDS Pathology Data Systems, Inc., Basel, Switzerland)].
Within a group, all body weights were within 20% of their mean. Animals selected for the study
receive a permanent identification number by ear tag and transponder and individual cage cards
also identified the study animals by individual numbers and group. The identifying numbers
assigned were unique within the study.
[0212] E. Experimental Design: All animal procedures were performed in an animal biosafety
level–2 facility in accordance with the protocols approved by the animal care and use committee
at IIT Research Institute. 6 male ferrets (Triple F Farms, Sayre PA), 4 months of age at the time
of study initiation were utilized for the study. Prior to infection, ferrets were monitored for 3
days to measure body weight and establish baseline body temperatures. Temperature readings
59
48204255.2
were recorded daily through a transponder (BioMedic data systems, Seaford, DE) implanted
subcutaneously in each ferret. Blood was collected prior to study initiation via the jugular vein,
and serum tested for influenza antibodies. Study animals free of influenza antibodies were
randomized and divided into two groups (3 ferrets/group) as shown in Table 15. A group of 3
ferrets was anesthetized and inoculated intranasally with a single dose of 316 µL at 1x107
TCID50 of M2KO(ΔTM) virus. A control group (3 ferrets) was inoculated with 316 µL at 1x107
TCID50 of A/Brisbane/10/2007 (H3N2). Ferrets were observed daily to monitor body weight,
body temperature and clinical symptoms. On Day 3 post-inoculation, ferrets (3 ferrets per group)
were euthanized and necropsied. The following tissue samples were collected: nasal turbinates,
trachea, lungs, kidneys, pancreas, olfactory bulbs, brains, livers, spleens, small and large
intestines. One part of the collected samples was fixed with buffered neutral formalin for
histological evaluation and the other part of the samples were stored at -65°C for virus titration.
[0213] F. Virus Inoculation: Ferrets were inoculated with either the M2KO(ΔTM) virus or wild
type A/Brisbane/10/2007 (H3N2) influenza A virus. A vial of frozen stock was thawed and
diluted to the appropriate concentration in phosphate buffered saline solution. Ferrets were
anesthetized with ketamine/xylazine and the virus dose administered intranasally in a volume of
316 µL for the M2KO(ΔTM) virus and 316 µL for the A/Brisbane/10/2007 (H3N2) virus. To
confirm the inoculation titer of the A/Brisbane/10/2007 (H3N2) virus, a TCID50 assay was
performed at IITRI on a portion of the prepared viral challenge solution. The viral titer assay was
60
48204255.2
performed according to Illinois Institute of Technology Research Institute (IITRI) Standard
Operating Procedures.
[0214] G. Moribundity/Mortality Observations: Following challenge, all animals were
observed twice daily for mortality or evidence of moribundity. Animals were observed for 3
days post-challenge. Animals were euthanized by overdose with Sodium Pentobarbital 150
mg/kg, administered intravenously.
[0215] H. Body Weights and Body Weight Change: Body weights of animals were recorded
upon receipt (random 10% sample), at randomization (Day -3 to 0), and daily after virus
inoculation.
[0216] I. Clinical Observations: The change in temperature (in degrees Celsius) was
determined daily for each ferret. Clinical signs of, inappetence, respiratory signs such as
dyspnea, sneezing, coughing, and rhinorrhea and level of activity was assessed daily. A scoring
system based on that described by Reuman, et al., “Assessment of signs of influenza illness in
the ferret model,” J. Virol, Methods 24:27-34 (1989), was used to assess the activity level as
follows: 0, alert and playful; 1, alert but playful only when stimulated; 2, alert but not playful
when stimulated; and 3, neither alert nor playful when stimulated. A relative inactivity index
(RII) was calculated as the mean score per group of ferrets per observation (day) over the
duration of the study.
[0217] J. Euthanasia: Study animals were euthanized by an intravenous dose of sodium
pentobarbital 150 mg/kg. Death was confirmed by absence of observable heartbeat and
respiration. Necropsies were performed on all study animals.
[0218] K. Necropsy: Nasal turbinates, trachea, lungs, kidneys, pancreas, olfactory bulbs, brain,
liver, spleen, small and large intestines were harvested. One portion of each tissue was fixed in
formalin and the other portion given to IITRI staff for freezing and storage. Tissue harvested for
titers are: right nasal turbinates, upper 1/3 of trachea, right cranial lung lobe, right kidney, right
61
48204255.2
arm of pancreas (near duodenum), right olfactory bulb, right brain, right lateral lobe of liver,
right half of spleen (end of spleen seen on opening the abdominal cavity), small intestine and
large intestine.
[0219] L. Histopathological analysis: Tissues were processed through to paraffin blocks,
sectioned at approximately 5-microns thickness, and stained with hematoxylin and eosin (H &
E).
[0220] M. Serum Collection: Pre-vaccination (Day -3) serum was collected from all ferrets.
Ferrets were anesthetized with a ketamine (25 mg/kg) and xylazine (2mg/kg) mixture. A sample
of blood (approximately 0.5-1.0 mL) was collected via the vena cava from each ferret and
processed for serum. Blood was collected into Serum Gel Z/1.1 tubes (Sarstedt Inc. Newton, NC)
and stored at room temperature for not more than 1 hour before collecting serum. Serum Gel
Z/1.1 tubes were centrifuged at 10,000xg for 3 minutes and the serum collected. Individual preinoculation serum samples were collected and two aliquots made from each sample. One aliquot
was tested prior to the initiation of the study to confirm ferrets are free of antibodies to influenza
A viruses and one aliquot of the serum stored at -65ºC.
[0221] N. Hemagglutination Inhibition (HI) Assay: Serum samples were treated with receptordestroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) to eliminate inhibitors of nonspecific
hemagglutination. RDE was reconstituted per the manufacturer’s instructions. Serum was
diluted 1:3 in RDE and incubated 18-20 hours in a 37ºC ± 2ºC water bath. After the addition of
an equal volume of 2.5% (v/v) sodium citrate, the samples were incubated in a 56 ± 2°C water
bath for 30 ± 5 minutes. 0.85% NaCl was added to each sample to a final serum dilution of 1:10
after the RDE treatment. The diluted samples were then diluted into four two-fold dilutions
(1:10 to 1:80) in duplicate in phosphate buffered saline (PBS) then incubated with 4
hemagglutinating units of A/Brisbane/10/2007 (H3N2) influenza A virus. After incubation,
0.5% chicken red blood cells were added to each sample and incubated. Presence or absence of
hemagglutination was then scored.
62
48204255.2
[0222] O. Virus Titers: The concentration of infectious virus in the pre- and post-challenge
virus inoculum samples was determined by TCID50 in Madin-Darby Canine Kidney (MDCK)
cells. Briefly, samples kept at -65°C were thawed and centrifuged to remove cellular debris.
The resulting supernatant were diluted 10-fold in triplicate in 96-well microtiter plates in
Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA) containing
Pencillin/Streptomycin, 0.1% Gentamicin, 3% NaCO3, 0.3% BSA fraction V (Sigma St. Louis,
MO), 1% MEM vitamin solution (Sigma) and 1% L-glutamine (Mediatech, Manassas, VA,
USA). After 10-fold serial dilutions were made, 100L was transferred into respective wells of a
96-well plate which contained a monolayer of MDCK cells. Plates were incubated at 37°C ± 2°C
in 5 ± 2% CO2 70% humidity. After 48 hours, the wells were observed for cytopathogenic effect
(CPE). Supernatant from each well (50 µl) was transferred to a 96 well plate and the
hemagglutination (HA) activity determined and recorded. The HA activity of the supernatant
was assessed by HA assay with 0.5% packed turkey red blood cells (cRBCs). TCID50 titers were
calculated using the method of Reed LJ and Muench H, “A simple method for estimating 50%
endpoints,” Am. J. Hygiene 27: 493-497 (1938).
[0223] P. Data Analysis: Body weights and body weight gains (losses) and changes in body
temperature were determined for each individual animal expressed as mean and standard
deviations of the mean for each test group.
RESULTS
[0224] After inoculation with either the M2KO(ΔTM) virus or A/Brisbane/10/2007 (H3N2)
influenza A virus, ferrets were monitored for survival and clinical signs of infection. Results are
presented in Table 16A and 16B. All ferrets survived infection with M2KO(ΔTM) virus and
A/Brisbane/10/2007 (H3N2). Ferrets inoculated with A/Brisbane/10/2007 presented respiratory
signs (sneezing) on Day 2 and 3. The relative inactivity index of ferrets inoculated with
A/Brisbane/10/2007 was 0.67; whereas ferrets inoculated with M2KO(ΔTM) showed no
reduction activity level with a relative inactivity index of 0.0.
63
48204255.2
[0225] Changes in body weight and temperature after virus inoculation are shown in Figure 20
and Figure 21. After inoculation with A/Brisbane/10/2007 (H3N2), a 2-3% loss of body weight
was observed on Day 2 post inoculation in all animals. Minimal to zero weight loss was
observed in ferrets inoculated with the M2KO(ΔTM) virus. One M2KO(ΔTM) inoculated ferret
exhibited weight loss on Day 2 post inoculation of 1%. Elevated body temperatures of 40.3-
40.7°C were observed in ferrets inoculated with A/Brisbane 10/2007 on Day 2 post inoculation.
Body temperatures returned to normal range by Day 3. Body temperatures for M2KO(ΔTM)
inoculated ferrets remained in normal range throughout the duration of the study. To determine if
the M2KO(ΔTM) virus would replicate in the respiratory tract or other organs and induce
pathology, tissues of ferrets were histologically examined on day 3 post inoculation and
compared to those from ferrets inoculated with A/Brisbane/10/2007. In ferrets inoculated with
A/Brisbane/10/2007, pathology was observed only in the nasal turbinates. Atrophy of respiratory
epithelium, infiltrates of neutrophils and edema were observed in the nasal turbinates. No
histopathological changes associated with viral infection were observed in ferrets inoculated with
the M2KO(ΔTM) virus. The concentrations of pre- and post-challenge virus dosing solutions
were 107.5 TCID50/mL and 107.75 TCID50/mL, respectively, indicating good stability of the
challenge material throughout administration.
64
48204255.2
Table 16B: M2KO(ΔTM) Does Not Replicate in Ferret Respiratory Organs Harvested On Day 3
Virus Titer (log pfu/g)
Brisbane/10 M2KO(ΔTM)
Nasal
Turbinates
.43 0
Lung 0 0
CONCLUSION
[0226] This example shows that by Day 3 post inoculation, the M2KO(ΔTM) virus does not
induce clinical signs of disease or histopathological changes associated with infection of wild
65
48204255.2
type virus. This shows that the M2KO(ΔTM) virus of the present technology is useful for
intranasal influenza vaccines.
Example 11: Immune Response and Protective Effects M2KO(ΔTM) Virus Relative To Other
Vaccines
[0227] Summary – This example demonstrates the immune response elicited by the
M2KO(ΔTM) vaccine and the protective effects of the vaccine in the ferret model. The
M2KO(ΔTM) virus was administered intransally to 12 male ferrets at a dose level of 1x107
TCID50. As a control, a second group of 12 male ferrets was administered the FM#6 virus
intranasally at a dose of 1x107 TCID50. A third group of ferrets was administered OPTI-MEM™
as a placebo control. A prime only or prime-boost vaccination regimen was utilized for each
treatment group. Ferrets receiving the prime-boost vaccination regimen were administered the
prime vaccine (Day 0) and the boost vaccination 28 days later (Day 28). Ferrets receiving only
the prime vaccine were administered a single vaccination on the same day as the booster vaccine
was given to the prime-boost ferrets (Day 28). Following each vaccination, ferrets were
observed for 14 days post inoculation for mortality, with body weights, body temperatures and
clinical signs measured daily. Nasal washes were collected from ferrets on days 1, 3, 5, 7 and 9
post-prime vaccination to look for viral shedding. Nasal washes and serum were collected
weekly from all ferrets post-vaccination to evaluate antibody levels over time.
[0228] All animals were challenged intranasally on Day 56 with 1x107 TCID50 of
A/Brisbane/10/2007 (H3N2). Following challenge, ferrets were monitored for 14 days post
inoculation for mortality, with body weights, body temperatures and clinical signs measured
daily. Nasal washes were collected on days 1, 3, 5, 7, 9 and 14 post challenge from ferrets in
each group for viral titers. Additionally, serum was collected post-challenge (day 70) from
surviving ferrets for analysis. Necropsy was performed on 3 ferrets per group 3 days post
challenge. Organs were collected for histopathology and viral titers.
66
48204255.2
[0229] No vaccine related adverse events were observed among the 5 groups. After challenge,
the placebo control group exhibited an increase in body temperature 2 days after challenge and a
reduction in weight. A reduction in weight was also observed in M2KO(ΔTM) and FM#6
vaccinated groups; however, the reduction was to less than that observed in the OPTI-MEM™
group. Activity levels were not reduced in any groups; however sneezing was observed in all
groups after challenge. Histopathological analysis revealed an increase in severity of mixed cell
infiltrates in the lung of vaccinated ferrets when compared to the lung infiltrates in the OPTIMEM™ control group. In the nasal turbinates, animals receiving a prime or prime plus boost
regimen of either M2KO(ΔTM) or FM#6 had lower severity of atrophy of respiratory epithelium
when compared to the OPTI-MEM™ control group. Vaccination with the M2KO(ΔTM) virus
appeared to provides similar protection against viral challenge as the FM#6 virus.
MATERIALS AND METHODS
[0230] A. Vaccine Material: The M2KO(ΔTM) virus is a recombinant virus which possesses
internal 6 genes of PR8 (nucleoprotein (NP), polymerase genes (PA, PB1, PB2), non-structural
(NS), matrix (M)), but which does not express functional M2 protein, as well as HA and NA
genes of Influenza A/Brisbane/10/2007-like A/Uruguay/716/2007(H3N2). The FM#6 virus is
clone #6 of the A/Uruguay/716/2007 (H3N2) influenza A virus from FluMist® (2009-2010
formula). The M2KO(ΔTM) virus and FM#6 virus were administered intranasally to the animals
in a 316 µL dose of 1x107 TCID50 (50% Tissue Culture Infectious Doses).
[0231] B. Test Article and Positive Control Dose Formulation: The M2KO(ΔTM) virus dosing
solution of 1x107 TCID50/mL per 316 µL was prepared by diluting 120 µL of 1x109 TCID50/mL
into 3.680 mL PBS. The FM#6 virus at a titer of 1x107 TCID50/mL per 316 µL was prepared by
diluting 120 µL of 1x109 TCID50/mL into 3.680 mL PBS.
[0232] C. Animals and Animal Care: Thirty-six male ferrets were purchased from Triple F
Farms and 30 of the ferrets were placed on study. Animals were approximately 4 months of age
67
48204255.2
at the time of study initiation. The animals were certified by the supplier to be healthy and free of
antibodies to infectious diseases. Upon arrival the animals were single housed in suspended wire
cages with slat bottoms, suspended over paper-lined waste pans. The animal room and cages had
been cleaned and sanitized prior to animal receipt, in accordance with accepted animal care
practices and relevant standard operating procedures. Certified Teklad Global Ferret Diet #2072
(Teklad Diets, Madison WI) and city of Chicago tap water were provided ad libitum and were
refreshed at least three time per week. Fluorescent lighting in the animal rooms was maintained
on a 12-hr light/dark cycle. Animal room temperature and relative humidity were within
respective protocol limits and ranged from 20.0 to 25.0°C and 30 to 63%, respectively, during
the study.
[0233] D. Animal Quarantine and Randomization: The ferrets were held in quarantine for
seven days prior to randomization and observed daily. Based on daily observations indicating
general good health of the animals the ferrets were released from quarantine for randomization
and testing. Following quarantine, ferrets were weighed and assigned to treatment groups using
a computerized randomization procedure based on body weights that produced similar group
mean values [ToxData® version 2.1.E.11 (PDS Pathology Data Systems, Inc., Basel,
Switzerland)]. Within a group, all body weights were within 20% of their mean. Animals
selected for the study receive a permanent identification number by ear tag and transponder and
individual cage cards also identified the study animals by individual numbers and group. The
identifying numbers assigned were unique within the study.
[0234] E. Experimental Design: To assess the M2KO(ΔTM) vaccine efficacy, ferrets were
immunized with M2KO(ΔTM) virus, cold adapted live attenuated virus (FM#6) or mock
immunized by medium (OPTI-MEM™ ). The animals body weight, body temperature and
clinical symptoms were monitored and immunological responses evaluated. 30 male ferrets
(Triple F Farms, Sayre PA), 4 months of age at the time of study initiation were utilized for the
study. All animal procedures were performed in an animal biosafety level–2 or biosafety level-3
68
48204255.2
facility in accordance with the protocols approved by the animal care and use committee at IIT
Research Institute. Prior to inoculation, ferrets were monitored for 3 days to measure body
weight and establish baseline body temperatures. Temperature readings were recorded daily
through a transponder (BioMedic data systems, Seaford, DE) implanted subcutaneously in each
ferret. Blood was collected prior to study initiation, and serum tested for influenza antibodies.
Only ferrets with HAI (hemagglutination inhibition) titers 40 to A/Brisbane/10/2007 (H3N2)
were considered seronegative and used in this study. Study animals were randomized and
divided into 5 groups (6 ferrets/group) as shown in Table 17. Two groups (1 & 3) received the
M2KO(ΔTM) virus and 2 groups (2 & 4) received the FM#6 virus. One group (5) was mock
immunized with OPTI-MEM™ . Within each vaccine group, ferrets were divided into two
vaccine regimens, six receiving a prime vaccination only (Prime only) and six receiving a prime
vaccination followed by a booster vaccine 28 days after prime vaccination (Prime/Boost).
Prime/Boost Groups: Ferrets were inoculated intranasally with a single dose of 316 µL of 1x107
TCID50 of M2KO(ΔTM) virus on days 0 and 28. Control groups were inoculated intranasally
with 316 µL of 1x107 TCID50 (same dose as M2KO(ΔTM)) of FM#6 or mock inoculated with
316 µL of OPTI-MEM™ on days 0 and 28. Ferret body temperatures, weights, and clinical
symptoms were monitored daily for 14 days post-inoculations. Nasal washes were collected from
all ferrets, including OPTI-MEM™ control group, on days 1, 3, 5, 7, 9 and 14 post prime
vaccination for virus titration in cells and on days 21 and 49 for antibody titration. Nasal wash
samples were kept at -65°C. Blood was collected prior to inoculation (day -3 to -5) and days 7,
14, 21 35, 42, and 49 and serum kept at -65°C until measurement of antibody titer by ELISA
and HI assay.
[0235] Prime only Groups: Ferrets were inoculated intranasally with a single dose of 316 µL
of 1x107 TCID50 of M2KO(ΔTM) virus on day 28. Control groups were inoculated intranasally
with 316 µL of 1x107 TCID50 (same dose as M2KO(ΔTM)) of FM#6 or mock inoculated with
316 µL of OPTI-MEM™ on day 28. Ferret body temperatures, weights, and clinical symptoms
were monitored daily for 14 days post-inoculation. Nasal washes were collected from all ferrets
69
48204255.2
on days 29, 31, 33, 35, 37, and 42 for virus titration in cells and on day 49 for antibody titration.
Nasal wash samples were kept at -65°C. Blood was collected prior to inoculation (day 23 to 25)
and days 35, 42, and 49 and serum was kept at -65°C until measurement of antibody titer by
ELISA and HAI assay. All ferrets were challenged with a dose of 316 µL of 1x107 TCID50 of
wild-type A/Brisbane/10/2007 (H3N2) influenza virus on day 56, 4 weeks after the prime/boost
vaccine was administered. Ferret body weight, body temperature and clinical symptoms were
monitored for 14 days after challenge and nasal washes and organs collected. Nasal washes were
collected from challenged ferrets on days 1, 3, 5, 7, 9, and 14 post-challenge (days 57, 59, 61, 63,
65, and 70) and the samples kept at -65°C for virus titration in cells. On Day 3 post-challenge
(day 59), the animals (3 animals per group, total 15 animals) were euthanized and the following
tissue samples collected: nasal turbinates, trachea, and lungs. One part of the collected samples
was fixed with buffered neutral formalin for histological evaluation and the other part of the
samples was stored at -65°C for virus titration. Blood was collected 14 days post-challenge (day
70) and all surviving animals were euthanized.
70
48204255.2
[0236] F. Virus Inoculation: Ferrets were inoculated with either the M2KO(ΔTM) virus or
FM#6 influenza A virus. A vial of frozen stock was thawed and diluted to the appropriate
concentration in phosphate buffered saline solution. Ferrets were anesthetized with
ketamine/xylazine and the virus dose administered intranasally in a volume of 316 µL for the
M2KO(ΔTM) virus and 316 µL for the FM#6 virus. To confirm the inoculation titer of the
M2KO(ΔTM) and FM#6 viruses, aliquots of the dosing solutions were collected prior to dosing
(pre-dose) and after dosing (post-dose). The aliquots were stored at -65°C for virus titration.
[0237] G. Challenge Virus: Influenza A virus, strain A/Brisbane/10/2007, serotype H3N2 was
used to challenge the ferrets. The virus was stored at approximately -65°C prior to use. The dose
level of challenge virus used was prepared at 1x107 TCID50 in a volume of 316 µL. A
quantitative viral infectivity assay, TCID50 assay was performed at IITRI on a portion of the
prepared viral challenge solution. The viral titer assay was performed according to IITRI
Standard Operating Procedures.
[0238] H. Moribundity/Mortality Observations: Following challenge, all animals were
observed twice daily for mortality or evidence of moribundity. Animals were observed for 14
days after vaccine inoculation and for 14 days after challenge.
[0239] I. Body Weights and Body Weight Change: Body weights were recorded within two
days of receipt and at randomization. All study animals were weighed prior to inoculation, daily
for 14 days following each vaccination and assessed daily for 14 days post challenge. Prior to
inoculation, ferrets were monitored for 3-5 days to measure establish baseline body temperatures.
Temperature readings were recorded daily for 14 days following each vaccination and recorded
daily for 14 days post challenge through a transponder (BioMedic data systems, Seaford, DE)
implanted subcutaneously in each ferret. The change in temperature (in degrees Celsius) was
calculated at each time point for each animal.
71
48204255.2
[0240] J. Clinical Observations: The change in temperature (in degrees Celsius) was
determined daily for each ferret. Clinical signs of, inappetence, respiratory signs such as
dyspnea, sneezing, coughing, and rhinorrhea and level of activity was assessed daily. A scoring
system based on that described by Reuman, et al., “Assessment of signs of influenza illness in
the ferret model,” J. Virol, Methods 24:27-34 (1989), was used to assess the activity level as
follows: 0, alert and playful; 1, alert but playful only when stimulated; 2, alert but not playful
when stimulated; and 3, neither alert nor playful when stimulated. A relative inactivity index
(RII) was calculated as the mean score per group of ferrets per observation (day) over the
duration of the study.
[0241] K. Survival Checks: Two survival checks were performed daily on all study animals
throughout the study. Both survival checks occurred simultaneously with the clinical
observations. The second check was performed later within the same day.
[0242] L. Nasal Washes: Ferrets were anesthetized with a ketamine (25 mg/kg) and xylazine
(2mg/kg) mixture, and 0.5 ml of sterile PBS containing penicillin (100 U/ml), streptomycin (100
µg/ml), and gentamicin (50 µg/ml) was injected into each nostril and collected in a specimen cup
when expelled by the ferret. The nasal wash was collected into a cryovial and the recovered
volume recorded.
[0243] M. Euthanasia: Study animals were euthanized by an intravenous dose of sodium
pentobarbital 150 mg/kg. Death was confirmed by absence of observable heartbeat and
respiration.
[0244] N. Necropsy: Necropsy was performed by Charles River Laboratories, Pathology
Associates (PAI). The PAI team was comprised of a supervising pathologist and two prosectors.
Nasal turbinates, trachea and lungs were harvested. One portion of each tissue was fixed in
formalin and the other portion given to IITRI staff for freezing and storage. Tissue harvested for
titers are: right nasal turbinates, upper 1/3 of trachea and right cranial lung lobe.
72
48204255.2
[0245] O. Histopathological analysis: Following each necropsy, tissues were transported to the
PAI Chicago facility. Upon receipt, partial tissues from all 15 ferrets were processed through to
paraffin blocks, sectioned at approximately 5-microns thickness, and stained with hematoxylin
and eosin (H & E). All paraffin H & E slides were evaluated microscopically.
[0246] P. Serum Collection: Pre-vaccination serum (days -3 to -5 for groups 3, 4, and 5, and
days 23 to 25 for groups 1 and 2) serum was collected from the ferrets. Post inoculation, serum
was collected on days 7, 14, 21, 35, 42, 49, and 70 from groups 3, 4, and 5. Serum was collected
on days 35, 42, 49, and 70 from groups 1 and 2. Ferrets were anesthetized with a ketamine (25
mg/kg) and xylazine (2mg/kg) mixture. A sample of blood (approximately 0.5-1.0 mL) was
collected via the vena cava from each ferret and processed for serum. Blood was collected into
Serum Gel Z/1.1 tubes (Sarstedt Inc. Newton, NC) and stored at room temperature for not more
than 1 hour before collecting serum. Serum Gel Z/1.1 tubes were centrifuged at 10,000xg for 3
minutes and the serum collected.
[0247] Q. Hemagglutination Inhibition (HI) Assay: Serum samples were treated with receptordestroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) to eliminate inhibitors of nonspecific
hemagglutination. RDE was reconstituted per the manufacturer’s instructions. Serum was
diluted 1:3 in RDE and incubated 18-20 hours in a 37ºC ± 2ºC water bath. After the addition of
an equal volume of 2.5% (v/v) sodium citrate, the samples were incubated in a 56 ± 2°C water
bath for 30 ± 5 minutes. 0.85% NaCl was added to each sample to a final serum dilution of 1:10
after the RDE treatment. The diluted samples were then diluted into four two-fold dilutions
(1:10 to 1:80) in duplicate in phosphate buffered saline (PBS) then incubated with 4
hemagglutinating units of A/Brisbane/10/2007 (H3N2) influenza A virus. After incubation,
0.5% avian red blood cells were added to each sample and incubated for 30 ± 5 minutes.
Presence or absence of hemagglutination was then scored.
[0248] R. Virus Titers: The concentration of infectious virus in the pre- and post-challenge
virus inoculum samples was determined by TCID50 assay in Madin-Darby Canine Kidney
73
48204255.2
(MDCK) cells. Briefly, samples kept at -65°C were thawed and centrifuged to remove cellular
debris. The resulting supernatant were diluted 10-fold in triplicate in 96-well microtiter plates in
Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA) containing
Pencillin/Streptomycin, 0.1% Gentamicin, 3% NaCO3, 0.3% BSA fraction V (Sigma St. Louis,
MO), 1% MEM vitamin solution (Sigma) and 1% L-glutamine (Mediatech, Manassas, VA,
USA). After 10-fold serial dilutions were made, 100 µL was transferred into respective wells of a
96-well plate which contained a monolayer of MDCK cells. Plates were incubated at 37°C ± 2°C
in 5 ± 2% CO2 70% humidity. After 48 hours, the wells were observed for cytopathogenic
effect (CPE). Supernatant from each well (50 µl) was transferred to a 96 well plate and the
hemagglutination (HA) activity determined and recorded. The HA activity of the supernatant
was assessed by HA assay with 0.5% packed turkey red blood cells (tRBCs). TCID50 titers were
calculated using the method of Reed LJ and Muench H, “A simple method for estimating 50%
endpoints,” Am. J. Hygiene 27: 493-497 (1938).
[0249] S. Data Analysis: Body weights and body weight gains (losses) and changes in body
temperature were determined for each individual animal expressed as mean and standard
deviations of the mean for each test group.
RESULTS
[0250] After intranasal vaccination with either the M2KO(ΔTM) virus or the FM#6 virus,
ferrets were monitored daily for clinical signs of infection. Nasal washes were collected after
prime vaccination to monitor viral shedding and serum collected to measure serum antibody
titers. Results are presented in Tables 18A, 18B, and 18C.
74
48204255.2
Table 18B: Virus Titers in Ferret Respiratory Organs After Challenge
Nasal Turbinates
(N=3, Log pfu/g)
Trachea
(N=3, Log pfu/g)
M2KO(ΔTM) prime only 5.23 ± 0.24 **
FluMist® prime only 5.53 ± 0.82 2.52 ± 1.73
M2KO(ΔTM) prime-boost 6.16 ± 1.17 1.37 ± 1.06
FluMist® prime-boost 6.24 ± 1.31 3.30 ± 1.96
**Not Detected
75
48204255.2
Table 18C: Mucosal IgA Responses in Ferret
α-HA ELISA IgA titers 14 days post-challenge:
Nasal
Wash Sera
M2KO(ΔTM)
prime only
14 Not Tested
FluMist® prime
only 29 Not Tested
[0251] All ferrets survived vaccination with M2KO(ΔTM) virus and FM#6 virus. After prime
vaccination, two ferrets inoculated with M2KO(ΔTM) virus presented respiratory signs
(sneezing) on Day 8. After boost vaccination, ferrets inoculated with the FM#6 virus presented
respiratory signs (sneezing) 7 days post vaccination. Sneezing was also observed in the OPTIMEM™ ferrets on day 4 post boost. After prime vaccination, the relative inactivity index of
ferrets inoculated with M2KO(ΔTM) virus and FM#6 virus was 0.07 and 0.27, respectively.
This reduction in activity was only observed in one group per virus after prime vaccination. After
boost vaccination no reduction in activity level was observed. Changes in body weight and
temperature after virus inoculation are shown in Figure 22 and Figure 23. No weight loss was
observed after vaccination; however, vaccination appeared to have an effect on weight gain.
After vaccination, body weights of OPTI-MEM™ control ferrets increased 20% during the 14
day observation whereas body weight gain of the M2KO(ΔTM) or FM#6 vaccinated ferrets
ranged from 6-15% after prime and 4-6% after boost. No increase in body temperature was
observed in any groups after vaccination. Changes in body weight and temperature after
challenge are shown in Figure 24 and Figure 25 and clinical signs summarized in Table 19.
76
48204255.2
a Clinical signs were observed for 3 days after virus inoculation. Except for lethargy,
findings for clinical signs are given as no. of ferrets with sign/total no. Respiratory signs
included sneezing.
b Determined twice daily for 3 days of observation based on the scoring system and was
calculated as the mean score per group of ferrets per observation (day) over the 3-day
period. The relative inactivity index before inoculation was 0.
[0252] After challenge with A/Brisbne/10/2007 (H3N2), a 2-4% loss of body weight was
observed on Day 2 post challenge in all animals. Throughout the 14 day observation period
animal body weights remained below their initial weight. OPTI-MEM™ ferrets lost the most
weight (8%). Weight loss among vaccinated ferrets was dependent on the vaccine regimen.
Ferrets receiving the prime only regimen of M2KO(ΔTM) or FM#6 lost a maximum of 5% and
4% respectively. Ferrets receiving a booster lost a maximum of 3% for the FM#6 group and 2%
for the M2KO(ΔTM) group. Elevated body temperatures post challenge were observed on Day 2
in OPTI-MEM™ ferrets and on Day 1 ferrets receiving the prime only regimens of
77
48204255.2
M2KO(ΔTM) or FM#6 (Figure 25). Body temperatures for ferrets receiving a booster remained
within normal range.
[0253] To determine if the vaccination would prevent replication of challenge virus in the
respiratory tract and reduce organ pathology tissues of challenged ferrets were histologically
examined on day 3 post inoculation. Changes in the lungs of animals receiving the
M2KO(ΔTM) prime only or prime/boost regimen were associated with increase in severity of
mixed cell infiltrates in the lung when compared to the OPTI-MEM™ group. Minor differences
in lung infiltrate incidences were observed between the M2KO(ΔTM) prime group and the
M2KO(ΔTM) prime/boost group. An increase in the severity of mixed cell infiltrates in the lung
was also seen in the FM#6 prime group and FM#6 prime/boost group when compared to the
OPTI-MEM™ group. A slight increase in severity in lung mixed cell infiltrates was observed in
the FM#6 prime/boost group over the FM#6 prime only group. In the nasal turbinates, animals
receiving the prime or prime/boost of the M2KO(ΔTM) virus had lower severity of atrophy of
respiratory epithelium when compared to the OPTI-MEM™ group. There were no differences in
atrophy of the nasal turbinates when comparing prime versus prime/boost M2KO(ΔTM) groups.
A slight increase in severity of atrophy of respiratory epithelium in animals receiving the FM#6
prime/boost regimen was observed versus animals FM#6 prime only regimen; the severity of
atrophy of respiratory epithelium in all FM#6 animals was lower than that seen in the OPTIMEM™ group. There was a decrease in incidence of neutrophilic infiltrates into the nasal cavity
(lumen) in the M2KO(ΔTM) prime and prime/boost groups compared to the OPTI-MEM™
group. Neutrophilic luminal infiltrates in the M2KO(ΔTM) prime only group was interpreted as
not different from the OPTI-MEM™ group. There was a slight increase in severity of luminal
neutrophilic infiltrates in the FM#6 prime only and prime/boost groups when compared to the
OPTI-MEM™ group. The concentrations of pre- and post-challenge virus dosing solutions were
107.83 TCID50/mL and 107.25 TCID50/mL, respectively, indicating good stability of the
challenge material throughout administration.
78
48204255.2
[0254] Figure 45 shows M2KO(ΔTM) and FluMist® virus replication in the ferret respiratory
tract.
[0255] Figure 46 shows M2KO(ΔTM) and FluMist® viral titers in nasal washes after
intranasal challenge with A/Brisbane/10/2007 (H3N2) virus.
[0256] Figure 47 shows IgG titers in ferrets following vaccination with M2KO(ΔTM) and
FluMist,® prime group only.
[0257] Figure 48 shows IgG titers in ferrets following vaccination with M2KO(ΔTM) and
FluMist,® prime-boost groups.
[0258] Figure 49 shows a summary of ELISA IgG titers in ferret sera from vaccination with
M2KO(ΔTM) or FluMist® to post-challenge.
CONCLUSION
[0259] This example shows that intranasal administration of the M2KO(ΔTM) virus was not
associated with any vaccine related adverse events (elevated body temperature, loss of weight or
clinical signs). These results show that the M2KO(ΔTM) virus of the present technology is
useful for use in an intranasal influenza vaccine.
Example 12: M2KO(ΔTM) Virus in Not Transmitted in the Ferret Model
[0260] Summary – This example demonstrates that the M2KO(ΔTM) virus is not transmitted
in the ferret model. The M2KO(ΔTM) virus was administered intransally to 3 female ferrets at a
dose level of 1x107 TCID50. As a control, a second group of 3 female ferrets was administered
the A/Brisbane/10/2007 (H3N2) virus intranasally at a dose of 1x107 TCID50. Twenty four hours
after inoculation, each donor ferret was introduced into a transmission chamber with two naive
ferrets (a direct contact and aerosol contact). Following inoculation, ferrets were observed for 14
days post inoculation for mortality, with body weights, body temperatures and clinical signs
79
48204255.2
measured daily. Nasal washes were collected from all inoculated donor ferrets on days 1, 3, 5, 7,
9 and from all contact (direct and aerosol) ferrets on days 2, 4, 6, 8, 10 to look for viral shedding.
Nasal washes and serum were collected from all ferrets at the inoculation of the study (Day 14)
to evaluate antibody levels. No clinical signs of infection were observed in the M2KO(ΔTM)
group; however, ferrets in the A/Brisbane/1 0/2007 (H3N2) group had weight loss, increased
body temperatures and were sneezing. After inoculation with Brisbane/1 0, the donor ferrets
exhibited an increase in body temperature 2 days after challenge and a reduction in weight.
Activity levels were not reduced in any groups. Ferrets in direct contact with the donor ferrets
showed progressive weight gain until day 4 post inoculation. A similar trend was observed in the
aerosol contact ferrets beginning on day 6 post inoculation. The loss in body weight in the
contact ferrets correlated with an increase in body temperature. Inoculation with the
M2KO(ΔTM) virus does not elicit clinical signs of infection in inoculated animals. Spread to
contact ferrets is unlikely.
MATERIALS AND METHODS
[0261] A. Vaccine Material: The M2KO(ΔTM) virus is a recombinant virus which possesses
internal 6 genes of PR8 (nucleoprotein (NP), polymerase genes (PA, PB1, PB2), non-structural
(NS), matrix (M)), but which does not express functional M2 protein, as well as HA and NA
genes of Influenza A/Brisbane/10/2007-like A/Uruguay/716/2007(H3N2). M2KO(ΔTM) virus
was administered intranasally to the animals in a 316 µL dose of 1xl07 TCID50 (50% Tissue
Culture Infectious Doses).
[0262] B. Test Article Dose Formulation: The M2KO(ΔTM) virus dosing solution of 1x107
TCID50/mL per 316 µL was prepared by diluting 45 of 1x109 TCID50/mL into 1.377 mL PBS.
[0263] C. Animals and Animal Care: 22 female ferrets were purchased from Triple F Farms
and 18 of the ferrets were placed on study. Animals were approximately 4 months of age at the
time of study initiation. The animals were certified by the supplier to be healthy and free of
80
48204255.2
antibodies to infectious diseases. Upon arrival the animals were single housed in suspended wire
cages with slat bottoms, suspended over paper-lined waste pans. The animal room and cages had
been cleaned and sanitized prior to animal receipt, in accordance with accepted animal care
practices and relevant standard operating procedures. Certified Teklad Global Ferret Diet #2072
(Teklad Diets, Madison WI) and city of Chicago tap water were provided ad libitum and were
refreshed at least once daily. Fluorescent lighting in the animal rooms was maintained on a 12-hr
light/dark cycle. Animal room temperature and relative humidity were within respective protocol
limits and ranged from 23.0 to 25.0°C and 36 to 50%, respectively, during the study.
[0264] D. Animal Quarantine and Randomization: The ferrets were held in quarantine for
seven days prior to randomization and observed daily. Based on daily observations indicating
general good health of the animals the ferrets were released from quarantine for randomization
and testing. Following quarantine, ferrets were weighed and assigned to treatment groups using
a computerized randomization procedure based on body weights that produced similar group
mean values [ToxData® version 2.l.E.11 (PDS Pathology Data Systems, Inc., Basel,
Switzerland)]. Within a group, all body weights were within 20% of their mean. Animals
selected for the study receive a permanent identification number by ear tag and transponder and
individual cage cards also identified the study animals by individual numbers and group. The
identifying numbers assigned were unique within the study.
[0265] E. Experimental Design: To assess the transmissibility of the M2KO(ΔTM) virus,
ferrets were inoculated with M2KO(ΔTM) virus or A/Brisbane/10/2007 (H3N2) virus. The
animals body weight, body temperature, clinical symptoms and viral shedding were monitored
and immunological responses evaluated. 18 female ferrets (Triple F Farms, Sayre PA), 4 months
of age at the time of study initiation were utilized for the study. All animal procedures were
performed in an animal biosafety level-2 or level 3 facility. Prior to inoculation, ferrets were
monitored for 3 days to measure body weight and establish baseline body temperatures.
Temperature readings were recorded daily through a transponder (BioMedic data systems,
81
48204255.2
Seaford, DE) implanted subcutaneously in each ferret. Blood was collected prior to study
initiation, and serum tested for influenza antibodies. Only ferrets with HI titers 40 to
A/Brisbane/1 0/2007 (H3N2) virus were considered seronegative and used in this study. Study
animals were randomized and divided into 2 groups (9 ferrets/group, 3/transmission chamber) as
shown in Table 20. Ferrets in group 1 (Chambers A-C) were assigned to receive the
M2KO(ΔTM) virus. Ferrets in group 2 (Chambers A-C) were assigned to receive the
A/Brisbane/1 0/2007 (H3N2) virus. Within each group, ferrets were divided into inoculated
donors or naive contacts.
[0266] Each group was housed in separate rooms, and individuals working with the animals
followed a strict work flow pattern to prevent cross contamination between the two groups In
each group, one donor ferret was inoculated intranasally with a single dose of 316 µL of 1x107
TCID50 of M2KO(ΔTM) (Group1) or 1x107 TCID50 of A/Brisbane/10/2007 (H3N2) virus
82
48204255.2
(Group 2). Twenty-four hours post inoculation; each donor was placed in the same cage with 1
naive ferret (direct contact), dual housed within a wire cage. An additional ferret (aerosol
contact) was placed in a separate adjacent wire cage (single housed) within the transmission
chamber separated from the donor's cage by a distance of 10-12 cm. Ferret body temperatures,
weights, and clinical symptoms were monitored daily for 14 days post-inoculation. Nasal washes
were collected from all inoculated donor ferrets on days 1, 3, 5, 7, 9 and from all contact (direct
and aerosol) ferrets on days 2, 4, 6, 8, 10 for virus titration in cells. Nasal washes were collected
from all ferrets on day 14 for antibody titration. Nasal wash samples were kept at -65°C.
[0267] F. Transmission Chambers: Each transmission chamber was 2 cubic meters. A
computerized air handling unit was used for HEPA filtration and to monitor and control
environmental conditions within the transmission chambers. To provide directional airflow,
HEPA-filtered air was supplied through an inlet port located at one end of the chamber, exited
through an outlet port at the opposite end the chamber, HEPA filtered and exhausted into the
room. Air exchange rate was 20 complete air changes per hour for each chamber, airflow was
maintained as <0.1 m/sec. Chambers were maintained at a negative pressure of -0.15 inches of
water. Ferrets were housed in wire cages with slat bottoms which were suspended over paperlined waste pans. Ferrets were either dual housed in 32x24x14 cages or single housed in
24x24x14 wire cages which were placed inside each HEPA-filtered transmission chamber.
[0268] G. Virus Inoculation: Ferrets were inoculated with the M2KO(ΔTM) virus. A vial of
frozen stock was thawed and diluted to the appropriate concentration in phosphate buffered
saline solution. Ferrets were anesthetized with ketamine/xylazine and the virus dose administered
intranasally in a volume of 316 µL for the M2KO(ΔTM). To confirm the inoculation titer of the
M2KO(ΔTM) virus, aliquots of the dosing solutions were collected prior to dosing (pre-dose)
and after dosing (post-dose). The aliquots were stored at 65°C for virus titration.
[0269] H. Challenge Virus: Influenza A virus, strain A/Brisbane/10/2007, serotype H3N2 was
used to inoculate the control ferrets. The virus was stored at approximately -65°C prior to use.
83
48204255.2
The dose level of challenge virus used was prepared at 1x1 07 TCID50 in a volume of 316 µL. A
quantitative viral infectivity assay, TCID50 assay was performed at IITRI on a portion of the
prepared viral challenge solution. The viral titer assay was performed according to IITRI
Standard Operating Procedures.
[0270] I. Moribundity/Mortality Observations: Following challenge, all animals were observed
twice daily for mortality or evidence of moribundity. Animals were observed for 14 days after
vaccine inoculation and for 14 days after challenge.
[0271] J. Body Weights and Body Weight Change: Body weights were recorded within two
days of receipt and at randomization. All study animals were weighed prior to inoculation, daily
for 14 days following each vaccination and assessed daily for 14 days post challenge. Prior to
inoculation, ferrets were monitored for 3-5 days to measure establish baseline body temperatures.
Temperature readings were recorded daily for 14 days following each vaccination and recorded
daily for 14 days post challenge through a transponder (BioMedic data systems, Seaford, DE)
implanted subcutaneously in each ferret. The change in temperature (in degrees Celsius) was
calculated at each time point for each animal.
[0272] K. Clinical Observations: The change in temperature (in degrees Celsius) was
determined daily for each ferret. Clinical signs of, inappetence, respiratory signs such as
dyspnea, sneezing, coughing, and rhinorrhea and level of activity was assessed daily. A scoring
system based on that described by Reuman, et al., “Assessment of signs of influenza illness in
the ferret model,” J. Virol, Methods 24:27-34 (1989), was used to assess the activity level as
follows: 0, alert and playful; 1, alert but playful only when stimulated; 2, alert but not playful
when stimulated; and 3, neither alert nor playful when stimulated. A relative inactivity index
(RII) was calculated as the mean score per group of ferrets per observation (day) over the
duration of the study.
84
48204255.2
[0273] L. Survival Checks: Two survival checks were performed daily on all study animals
throughout the study. Both survival checks occurred simultaneously with the clinical
observations. The second check was performed later within the same day.
[0274] M. Nasal Washes: Ferrets were anesthetized with a ketamine (25 mg/kg) and xylazine
(2mg/kg) mixture, and 0.5 ml of sterile PBS containing penicillin (100 U/ml), streptomycin (100
and gentamicin (50 was injected into each nostril and collected in a specimen cup when expelled
by the ferret.
[0275] N. Euthanasia: Study animals were euthanized by an intravenous dose of sodium
pentobarbital 150 mg/kg. Death was confirmed by absence of observable heartbeat and
respiration. Necropsies were performed on all study animals.
[0276] O. Serum Collection: Pre-vaccination serum (days -3 to -5) and post inoculation serum
(day 14) was collected from all ferrets. Ferrets were anesthetized with a ketamine (25 mg/kg) and
xylazine (2mg/kg) mixture. A sample of blood (approximately 0.5-1.0 mL) was collected via the
vena cava from each ferret and processed for serum. Blood was collected into Serum Gel Z/1.1
tubes (Sarstedt Inc. Newton, NC) and stored at room temperature for not more than 1 hour before
collecting serum. Serum Gel Z/1.1 tubes were centrifuged at 10,000xg for 3 minutes and the
serum collected. Individual pre-inoculation serum samples were collected and two aliquots made
from each sample. One aliquot was tested prior to the initiation of the study to confirm ferrets are
free of antibodies to influenza A viruses and one aliquot of the serum stored at -65°C.
[0277] P. Hemagglutination Inhibition (HI) Assay: Serum samples were treated with receptordestroying enzyme (RDE) (Denka Seiken, Tokyo, Japan) to eliminate inhibitors of nonspecific
hemagglutination. RDE was reconstituted per the manufacturer's instructions. Serum was diluted
1:3 in RDE and incubated 18-20 hours in a 37°C ± 2°C water bath. After the addition of an equal
volume of 2.5% (v/v) sodium citrate, the samples were incubated in a 56± 2°C water bath for 30
± 5 minutes. 0.85% NaCI was added to each sample to a final serum dilution of 1:10 after the
85
48204255.2
RDE treatment. The diluted samples were then diluted into four two-fold dilutions (1:10 to 1:80)
in duplicate in phosphate buffered saline (PBS) then incubated with 4 hemagglutinating units of
A/Brisbane/10/2007 (H3N2) influenza A virus. After incubation, 0.5% avian red blood cells
were added to each sample and incubated for 30 ± 5 minutes. Presence or absence of
hemagglutination was then scored.
[0278] Q. Virus Titers: The concentration of infectious virus in the pre-and post-challenge
virus inoculum samples was determined by TCID50 assay in Madin-Darby Canine Kidney
(MDCK) cells. Briefly, samples kept at -65°C were thawed and centrifuged to remove cellular
debris. The resulting supernatant were diluted 10-fold in triplicate in 96-well microtiter plates in
Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA) containing
Pencillin/Streptomycin, 0.1% Gentamicin, 3% NaCO3, 0.3% BSA fraction V (Sigma St. Louis,
MO), 1% MEM vitamin solution (Sigma) and 1% L-glutamine (Mediatech, Manassas, VA,
USA). After 10-fold serial dilutions were made, 1 OOflL was transferred into respective wells of
a 96-well plate which contained a monolayer of MDCK cells. Plates were incubated at 37°C ±
2°C in 5 ± 2% CO2 70% humidity. After 48 hours, the wells were observed for cytopathogenic
effect (CPE). Supernatant from each well (50 µl) was transferred to a 96 well plate and the
hemagglutination (HA) activity determined and recorded. The HA activity of the supernatant
was assessed by HA assay with 0.5% packed turkey red blood cells (tRBCs). TCID50 titers were
calculated using the method of Reed LJ and Muench H, “A simple method for estimating 50%
endpoints,” Am. J. Hygiene 27: 493-497 (1938).
[0279] R. Data Analysis: Body weights and body weight gains (losses) and changes in body
temperature were determined for each individual animal expressed as mean and standard
deviations of the mean for each test group.
86
48204255.2
RESULTS
[0280] After inoculation of donor ferrets with either the M2KO(ΔTM) virus or the
A/Brisbane/1 0/2007 (H3N2) influenza A virus donor ferrets were introduced into transmission
chambers containing naive contact ferrets. Ferrets were monitored daily for clinical signs of
infection, nasal washes were collected to monitor viral shedding and serum collected to measure
serum antibody titers. All ferrets survived inoculation with M2KO(ΔTM) virus and
A/Brisbane/10/2007 (Table 21). No clinical signs of disease were observed in ferrets in the
M2KO(ΔTM) group. Two of the three donor ferrets inoculated with A/Brisbane/10/2007 virus
presented respiratory signs (sneezing) on Day 6 and 8. Direct contact ferrets in all chambers
presented with sneezing on Day 8. No sneezing was observed in the aerosol contact ferrets. A
reduction in activity level was not observed.
87
48204255.2
[0281] Changes in body weight and temperature after virus inoculation are shown in Figure 26
and Figure 27. No significant weight loss was observed after inoculation with the M2KO(ΔTM)
virus. The aerosol contacts averaged a 1% loss in weight on day; however, it is unlikely this due
to exposure to virus. Body weights of ferrets in the M2KO(ΔTM) virus increased was 9% for
donor ferrets and 10-11% for contact ferrets during the 14 day observation (Figure 26A). Body
weight gain of the A/Brisbane/10/2007 was only 3% for donor ferrets and 6-8% for contact
ferrets indicating a viral infection (Figure 26B). In the M2KO(ΔTM) group, body temperatures
remained with in normal levels with the exception of Day 3 post infection (Figure 27A). Body
temperatures were lower than normal for the aerosol contact ferrets. This was attributed to faulty
or failing temperature transponders, temperatures were recorded within normal range throughout
the rest of the study. Elevated body temperatures were observed on Day 2 in A/Brisbane/10/2007
donor ferrets and on Day 7 for aerosol contacts (Figure 27B). The concentrations of pre-and
post-challenge virus dosing solution were 107.50 TCID50/mL and 107.25 TCID50/mL, respectively,
indicating good stability of the challenge material throughout administration.
[0282] Figure 50 shows viral titers in nasal washes from ferrets in a virus transmission study.
The data shows that M2KO(ΔTM) virus does not transmit (no virus detected), whereas the
control Brisb/10 virus is transmitted.
CONCLUSION
[0283] This example shows that ferrets inoculated with the A/Brisbane/10/2007 virus exhibited
clinical signs of infection (sneezing, loss in body weight and a transient elevated body
temperature), whereas ferrets inoculated with the M2KO(ΔTM) virus showed no clinical signs of
disease. Therefore, inoculation of donor ferrets with the M2KO(ΔTM) did not appear to cause an
infection or transmit virus via contact or via aerosol. These findings show that the M2KO(ΔTM)
virus of the present technology is useful for intranasal influenza vaccines.
88
48204255.2
Example 13. M2KO(ΔTM) Virus Elicits Both Humoral and Mucosal Immune Responses in
Mice.
[0284] This examples demonstrates that the M2KO(ΔTM) virus elicits both humoral and
mucosal immune responses in mice. The immunogenicity of M2KO(ΔTM) was evaluated in
mice and compared to the immune responses generated by other modes of vaccination. An
immunogenicity study was performed containing the following groups as outline in Table 22: 1.
M2KO(ΔTM) virus, 2. PR8 virus (10 pfu), live vaccine representative, 3. Inactivated PR8 virus
(Charles River Laboratories, Wilmington, MA), 1 µg, intranasal (IN) 4. Inactivated PR8 virus, 1
µg, intramuscular (IM), or PBS only.
Table 22: Vaccine Groups in Immunogenicity Study
Immunogen Route of Delivery Dose Rationale
M2KO(ΔTM) virus Intranasal 1x104 pfu
Comprises
M2KO(ΔTM) (SEQ
ID NO:1) Mutation
PR8 virus Intranasal 10 pfu
Represents the
immune responses
associated with a
natural infection
and/or live flu vaccine
Inactivated PR8,
whole virus Intranasal 1 µg
Demonstrates baseline
response generated by
killed flu virus
delivered intranasally
Inactivated PR8,
whole virus Intramuscular 1 µg
Standard delivery
route for traditional
inactivated flu vaccine
89
48204255.2
[0285] To test the immunogenicity of M2KO(ΔTM) virus, mice were intranasally inoculated
with 1.2x104 pfu of M2KO(ΔTM), 10 pfu of wild-type PR8, 1 µg of inactivated whole PR8
(Charles River Laboratories, Wilmington, MA), or PBS as control, along with a group
intramuscularly administered 1 µg of inactivated whole PR8. Three weeks after the
immunization, serum and trachea-lung washes were collected from mice and anti-PR8
immunoglobulin G (IgG) and IgA levels were measured by enzyme linked immunosorbent assay
(ELISA). Briefly, ELISA plates were coated by whole inactivated PR8, blocked by bovine serum
albumin (BSA), and samples were applied. Mouse IgG and IgA antibodies were detected by
horseradish peroxidase labeled anti-mouse IgG- and IgA- goat antibodies (KPL, Inc.,
Gaithersburg, MD) and SureBlue TMB (KPL, Inc.) substrate.
[0286] As expected, mice in the immunized groups showed significant elevation of anti-PR8
antibodies in serum and trachea-lung wash compare to the PBS only group (Figure 28). AntiPR8 IgG levels in sera for M2KO(ΔTM) virus are higher than the inactivated PR8 groups and
similar to live PR8 virus. More importantly anti-PR8 IgA antibodies were present only in the
PR8 and M2KO(ΔTM) immunized mice in both sera and trachea-lung washes. These data
suggest that M2KO(ΔTM) virus elicits significant humoral and mucosal immune response in
mice.
Example 14 M2KO(ΔTM) Virus Protects Mice From Lethal Homosubtypic and Heterosubtypic
Challenge.
[0287] This example demonstrates that the M2KO(ΔTM) virus protects mice from lethal
homosubtypic and heterosubtypic challenge. The protective efficacy M2KO(ΔTM) virus was
evaluated by challenging the immunized mice with lethal doses of the wild-type PR8 (H1N1;
homosubtypic challenge) or mouse-adapted influenza A/Aichi/2/68 (Aichi; H3N2;
heterosubtypic challenge) six weeks post-immunization. None of the mice immunized with
either M2KO(ΔTM) or 10 pfu of PR8 and subsequently challenged by wild-type PR8 showed
any clinical symptoms including weight loss (Figure 29A). In contrast, naive PBS mice died or
90
48204255.2
were euthanized due to greater than 20% weight loss by day 5. Virus replication in the
respiratory tracts of challenged mice was determined on day 3 post-challenge by TCID50 assay in
MDCK cells. As shown in Figure 30A, no virus was detected (limit of detection
102.75TCID50/organ) in the lungs of M2KO(ΔTM) or PR8 immunized mice indicating that
M2KO(ΔTM) provided sterile immunity similar to PR8 infection. In contrast, challenge virus
was recovered from the inactivated PR8 and PBS groups.
[0288] For heterosubtypic challenge, mice were challenged by Aichi (H3N2). M2KO(ΔTM)
and wild-type PR8 immunized mice survived challenge whereas mice that received inactivated
PR8 or PBS succumbed to infection (Figure 29). Virus titers in mouse respiratory tracts on day
3 post-challenge did not show significant reduction in M2KO(ΔTM) -vaccinated mice compared
to mice in other groups (Figure 30). These results suggest that the cross-protection observed
against Aichi challenge may in part be due to T-cell mediated immune responses induced by the
M2KO(ΔTM) vaccine. Hemagglutination inhibition (HI) antibodies to Aichi were not detectable
(less than 1:40) in post-challenge sera from challenged mice suggesting that protection was not
mediated by neutralizing antibodies.
[0289] The M2KO(ΔTM) virus stimulates both humoral and cellular immune responses and
confers protective immunity to animals against lethal homo- and hetero-subtypic challenge as
summarized in Table 23.
Table 23 Protection After Homosubtypic (H1N1) and Heterosubtypic (H3N2) Influenza
Challenge
Survival (%)
Vaccine Group PR8 (H1N1) Challenge Aichi (H3N2) Chalenge
M2KO(ΔTM) 100% 100%
PR8 100% 100%
91
48204255.2
Inactivated PR8, IN 100% 0%
Inactivated PR8, IM 100% 20%
PBS 0% 0%
Example 15 M2KO(ΔTM) Vaccine Compared to Fluzone® and FluMist®
[0290] This example demonstrates the efficacy of the M2KO(ΔTM) virus compared to ive
attenuated virus (FluMist®), Fluzone® inactivated flu vaccine. Mice were immunized with
M2KO(ΔTM) virus, cold adapted live attenuated virus (FluMist®), Fluzone® inactivated flu
vaccine or mock immunized by PBS. M2KO(ΔTM)-H3 virus was constructed by inserting the
HA and NA coding sequences of Influenza A/Brisbane/10/2007-like,
A/Uruguay/716/2007(H3N2) in to the M2KO(ΔTM) backbone (SEQ ID NO:1). FluMist®-H3,
internal genes from the cold-adapted A/AA/6/60 backbone, containing the HA and NA genes of
Influenza A/Brisbane/10/2007-like, A/Uruguay/716/2007(H3N2) was plaque purified from the
2009/2010 trivalent vaccine formulation. Fluzone® 2009/2010 formulation was used directly as
the trivalent formulation.
[0291] Sera was obtained on days 7, 14, 21 post-immunization to compare the kinetics of
antibody response by ELISA (Figure 31). M2KO(ΔTM)-H3 virus, a replication deficient virus,
developed antibodies earlier than FluMist®-H3, a live flu virus vaccine that undergoes multicycle replication in an attenuated manner. The inactivated vaccine Fluzone® had the highest
antibody titers in sera as it is a concentrated presentation of antigen.
[0292] The presence of anti-HA mucosal antibody in sera, lung wash, and nasal turbinates was
evaluated by ELISA. M2KO(ΔTM)-H3 and FluMist,® the two live flu vaccines, had higher IgA
in the respiratory tract than the inactivated vaccine Fluzone® . (Figure 32)
92
48204255.2
Example 16: Comparison of Protection and Immunogenicity Elicited By Live Viruses.
[0293] Six-week-old female BALB/c mice, anesthetized with isoflurane, were infected
intranasally on days 0 and 28 with 106 TCID50/50µl of M2KO(ΔTM)-H3 (described above),
FluMist® (2009-2010) (H3N2) IVR-147 (PR8xBrisbane/10/2007). IVR-147 is the wild-type
version of the M2KO(ΔTM) virus; i.e. contains a functional M2 protein. Mock-infected control
mice received 50 μl PBS instead of virus. Serum was collected weekly from all the mice and
analyzed for the presence of anti-HA antibodies by ELISA. As shown in Figure 33,
M2KO(ΔTM) virus and IVR-147 generated higher antibody levels with rapid kinetics compared
to FluMist®.
[0294] Body weights of animals were monitored for 14 days after infection. Vaccinated mice
did not lose any weight. On day 21 post-boost, 3 mice per group were euthanized and their
trachea-lung washes, nasal washes, and sera were collected for antibody titer determinations
(Figure 34). M2KO(ΔTM) induced both humoral and mucosal antibodies to similar levels as
FluMist® and IVR-147 in sera and respiratory tract.
[0295] Mice were intranasally challenged with 40MLD50 of A/Aichi/2/68 virus six weeks
post-boost. Mice were observed for loss of body weight and survival for 14 days (Figure 35).
M2KO(ΔTM) protected mice from lethal Aichi challenge as indicated by less body weight loss
(Panel A) and 100% survival (Panel B) in contrast to FluMist®. On day 3 post-challenge, 3 mice
per group were euthanized and their lungs and nasal turbinates were collected for virus titer
determinations (Table 24). M2KO(ΔTM) controlled the challenge virus better than FluMist® as
shown in Table 24.
93
48204255.2
Table 24. Challenge virus titers in respiratory tract.
Example 17: Generation of an M2KO(ΔTM) Vaccine Against Highly Pathogenic Avian H5n1
Influenza Virus
[0296] Summary: M2KO(ΔTM) is an influenza virus that lacks expression of a functional M2
protein. The M2 protein is crucial for initiation of influenza viral infection and for efficient viral
RNA incorporation into progeny virions. M2KO(ΔTM) can enter cells and express viral proteins
but cannot make infectious progeny viruses due to deletion of the M2 gene. M2KO(ΔTM) is
produced in permissive M2 protein expressing cells but not in non-permissive wild-type cells.
M2KO(ΔTM) elicits both mucosal and humoral immunity in mice and protects from both homoand hetero-subtypic lethal challenge.
[0297] The H5N1 M2KO(ΔTM) virus contains the HA (avirulent) and NA genes of
A/Vietnam/1203/2004 on the M2KO(ΔTM) backbone. By “M2KO(ΔTM) backbone” is meant
the sequence of PR8 comprising the M2KO(ΔTM) (SEQ ID NO:1) mutation. The
A/Vietnam/1203/2004 HA (avirulent) (SEQ ID NO: 24) and NA (SEQ ID NO: 25) sequences
used are shown below.
Lung
(Log TCID50/g)
Nasal Turbinate
(Log TCID50/g)
Mean ± SD Mean ± SD
M2KO H3 7.05 ± 0.14 4.37 ± 1.01
FluMist H3 7.32 ± 0.38 6.83 ± 1.50
IVR 147 7.08 ± 0.14 4.87 ± 0.14
PBS 7.95 ± 0.63 6.25 ± 0.29
94
48204255.2
>Avirulent VN1203 HA ORF + PR8 non-coding
AGCAAAAGCAGGGGAAAATAAAAACAACCAAAATGGAGAAAATAGTGCTTCTTTTTGCAATAGTCAGTCTTGTTAAAAGT
GATCAGATTTGCATTGGTTACCATGCAAACAACTCGACAGAGCAGGTTGACACAATAATGGAAAAGAACGTTACTGTTAC
ACATGCCCAAGACATACTGGAAAAGAAACACAACGGGAAGCTCTGCGATCTAGATGGAGTGAAGCCTCTAATTTTGAGAG
ATTGTAGCGTAGCTGGATGGCTCCTCGGAAACCCAATGTGTGACGAATTCATCAATGTGCCGGAATGGTCTTACATAGTG
GAGAAGGCCAATCCAGTCAATGACCTCTGTTACCCAGGGGATTTCAATGACTATGAAGAATTGAAACACCTATTGAGCAG
AATAAACCATTTTGAGAAAATTCAGATCATCCCCAAAAGTTCTTGGTCCAGTCATGAAGCCTCATTAGGGGTGAGCTCAG
CATGTCCATACCAGGGAAAGTCCTCCTTTTTCAGAAATGTGGTATGGCTTATCAAAAAGAACAGTACATACCCAACAATA
AAGAGGAGCTACAATAATACCAACCAAGAAGATCTTTTGGTACTGTGGGGGATTCACCATCCTAATGATGCGGCAGAGCA
GACAAAGCTCTATCAAAACCCAACCACCTATATTTCCGTTGGGACATCAACACTAAACCAGAGATTGGTACCAAGAATAG
CTACTAGATCCAAAGTAAACGGGCAAAGTGGAAGGATGGAGTTCTTCTGGACAATTTTAAAGCCGAATGATGCAATCAAC
TTCGAGAGTAATGGAAATTTCATTGCTCCAGAATATGCATACAAAATTGTCAAGAAAGGGGACTCAACAATTATGAAAAG
TGAATTGGAATATGGTAACTGCAACACCAAGTGTCAAACTCCAATGGGGGCGATAAACTCTAGCATGCCATTCCACAATA
TACACCCTCTCACCATTGGGGAATGCCCCAAATATGTGAAATCAAACAGATTAGTCCTTGCGACTGGGCTCAGAAATAGC
CCTCAAAGAGAGACTAGAGGATTATTTGGAGCTATAGCAGGTTTTATAGAGGGAGGATGGCAGGGAATGGTAGATGGTTG
GTATGGGTACCACCATAGCAATGAGCAGGGGAGTGGGTACGCTGCAGACAAAGAATCCACTCAAAAGGCAATAGATGGAG
TCACCAATAAGGTCAACTCGATCATTGACAAAATGAACACTCAGTTTGAGGCCGTTGGAAGGGAATTTAACAACTTAGAA
AGGAGAATAGAGAATTTAAACAAGAAGATGGAAGACGGGTTCCTAGATGTCTGGACTTATAATGCTGAACTTCTGGTTCT
CATGGAAAATGAGAGAACTCTAGACTTTCATGACTCAAATGTCAAGAACCTTTACGACAAGGTCCGACTACAGCTTAGGG
ATAATGCAAAGGAGCTGGGTAACGGTTGTTTCGAGTTCTATCATAAATGTGATAATGAATGTATGGAAAGTGTAAGAAAT
GGAACGTATGACTACCCGCAGTATTCAGAAGAAGCGAGACTAAAAAGAGAGGAAATAAGTGGAGTAAAATTGGAATCAAT
AGGAATTTACCAAATACTGTCAATTTATTCTACAGTGGCGAGTTCCCTAGCACTGGCAATCATGGTAGCTGGTCTATCCT
TATGGATGTGCTCCAATGGGTCGTTACAATGCAGAATTTGCATTTAAGATTAGAATTTCAGAGATATGAGGAAAAACACC
CTTGTTTCTACT
>VN1203 NA ORF + PR8 non-coding
AGCAAAAGCAGGGGTTTAAAATGAATCCAAATCAGAAGATAATAACCATCGGATCAATCTGTATGGTAACTGGAATAGTT
AGCTTAATGTTACAAATTGGGAACATGATCTCAATATGGGTCAGTCATTCAATTCACACAGGGAATCAACACCAATCTGA
ACCAATCAGCAATACTAATTTTCTTACTGAGAAAGCTGTGGCTTCAGTAAAATTAGCGGGCAATTCATCTCTTTGCCCCA
TTAACGGATGGGCTGTATACAGTAAGGACAACAGTATAAGGATCGGTTCCAAGGGGGATGTGTTTGTTATAAGAGAGCCG
TTCATCTCATGCTCCCACTTGGAATGCAGAACTTTCTTTTTGACTCAGGGAGCCTTGCTGAATGACAAGCACTCCAATGG
GACTGTCAAAGACAGAAGCCCTCACAGAACATTAATGAGTTGTCCTGTGGGTGAGGCTCCCTCCCCATATAACTCAAGGT
TTGAGTCTGTTGCTTGGTCAGCAAGTGCTTGCCATGATGGCACCAGTTGGTTGACGATTGGAATTTCTGGCCCAGACAAT
GGGGCTGTGGCTGTATTGAAATACAATGGCATAATAACAGACACTATCAAGAGTTGGAGGAACAACATACTGAGAACTCA
AGAGTCTGAATGTGCATGTGTAAATGGCTCTTGCTTTACTGTAATGACTGACGGACCAAGTAATGGTCAGGCATCACATA
AGATCTTCAAAATGGAAAAAGGGAAAGTGGTTAAATCAGTCGAATTGGATGCTCCTAATTATCACTATGAGGAATGCTCC
TGTTATCCTAATGCCGGAGAAATCACATGTGTGTGCAGGGATAATTGGCATGGCTCAAATCGGCCATGGGTATCTTTCAA
TCAAAATTTGGAGTATCAAATAGGATATATATGCAGTGGAGTTTTCGGAGACAATCCACGCCCCAATGATGGAACAGGTA
GTTGTGGTCCGGTGTCCTCTAACGGGGCATATGGGGTAAAAGGGTTTTCATTTAAATACGGCAATGGTGTCTGGATCGGG
AGAACCAAAAGCACTAATTCCAGGAGCGGCTTTGAAATGATTTGGGATCCAAATGGGTGGACTGAAACGGACAGTAGCTT
TTCAGTGAAACAAGATATCGTAGCAATAACTGATTGGTCAGGATATAGCGGGAGTTTTGTCCAGCATCCAGAACTGACAG
GACTAGATTGCATAAGACCTTGTTTCTGGGTTGAGTTGATCAGAGGGCGGCCCAAAGAGAGCACAATTTGGACTAGTGGG
AGCAGCATATCTTTTTGTGGTGTAAATAGTGACACTGTGGGTTGGTCTTGGCCAGACGGTGCCGAGTTGCCATTCACCAT
TGACAAGTAGTCTGTTCAAAAAACTCCTTGTTTCTACT
95
48204255.2
[0298] Generation of H5N1 M2KO(ΔTM): The avirulent HA and NA of
A/Vietnam/1203/2004 (H5N1) were chemically synthesized by GeneArt® Gene Synthesis based
on the CDC sequences for each gene (CDC ID: 2004706280, Accession Numbers: EF541467
and EF541403). The sequences of the constructs were confirmed and sub-cloned into
appropriate vectors to allow for the generation of seed virus using standard protocols.
[0299] M2KO(ΔTM) VN1203avHA,NA (H5N1 M2KO(ΔTM)) virus was amplified in M2CK
cells (MDCK cells stably expressing the M2 protein), the supernatant clarified of cell debris and
concentrated 100-fold by Centricon Plus-70 (Millipore). This virus was used as the immunogen
in the mice study.
[0300] Mouse Study Design: Mice (7-8 weeks old, female BALB/c) were intranasally
inoculated with H5N1 M2KO(ΔTM) (106 TCID50/mouse), M2KO(ΔTM) CA07HA, NA (106
TCID50/mouse) or VN1203 protein (1.5 µg) administered intramuscularly. Body weight and
clinical symptoms were observed for 14 days post-inoculation. Sera was collected on days 7, 14,
21 post-inoculation. Mice were boosted on day 28 with a new prime only group initiated at the
same time.
[0301] Boost immunization and ‘prime only’ groups: On day 28 the mice previously
inoculated with H5N1 M2KO(ΔTM) were boosted with a second immunization of 106
pfu/mouse. At the same time the ‘prime only’ groups were given their first dose. Weight loss
was followed for all groups following the day 28 inoculation. The mice that received a boost
dose of M2KO(ΔTM) vaccine lost at most 5% of their body weight. The ‘prime only’ group lost
up to 10% of their body weight.
96
48204255.2
Table 25. Vaccine groups in mice study
Group1 Immunogen Doses
Route of
Administration
Challenge Virus
1 H5N1 M2KO(ΔTM) 2 Intranasal
Challenged 5
months postimmunization
with 20 MLD50
A/VN/1203/2004
2 H5N1 M2KO(ΔTM) 1 Intranasal
3 H1N1pdm
M2KO(ΔTM)
2
Intranasal
4
H5 HA VN1203
protein
2
Intramuscular
Naïve (OPTIMEM™ )
2
Intranasal
6 H5N1 M2KO(ΔTM) 1 Intranasal Challenged 4
weeks postimmunization
with 20 MLD50
A/VN/1203/2004
7
Naïve (OPTIMEM™ ) 1
Intranasal
mice/group for survival assessment after challenge
[0302] H5N1 M2KO(ΔTM) elicits IgG antibody titers against HA: Sera was obtained from
mice on day 7, 14, 21 post-inoculation and analyzed by ELISA for antibodies against the
hemagglutinin. M2KO(ΔTM) generated at least 100 fold higher titers than H5 HA protein
97
48204255.2
(Figure 36). Mice were boosted on day 28 and sera was obtained a week later (day 35). The
M2KO(ΔTM) titers were boosted 130 fold, whereas the HA protein only boosted 13 fold. The
first week bleed at day 35 for the M2KO(ΔTM) prime only groups demonstrated high IgG titers
as the first week of the prime-boost groups.
[0303] Mice were challenged with a lethal dose of Vietnam/1203/2004 virus (20 MLD50). All
H5N1 M2KO(ΔTM) vaccinated (prime only and prime-boost) mice survived (Figures 54 and
55). The high survival rate of mice challenged 5 months post-immunization suggests that the
H5N1 M2KO(ΔTM) vaccine primes memory responses. Mice challenged 4 weeks postimmunization had received only one dose of vaccine, indicating that the M2KO(ΔTM) vaccine
stimulates a strong immune response. H1N1pdm M2KO(ΔTM) immunized mice also survived
H5N1 challenge after 5 months indicating that M2KO(ΔTM) primes cross-reactive immune
responses that provide protection against heterologous challenge.
Example 18: H1N1pdm: FluMist® CA07 vs M2KO(ΔTM) CA07
[0304] The HA and NA cDNA clones of A/California/07/2009 (CA07) (H1N1pdm) were
generated by standard molecular biology protocols. The sequences of the constructs were
confirmed and sub-cloned into appropriate vectors to allow for the generation of seed
M2KO(ΔTM) virus and M2WTCA07/PR8 virus using standard protocols. FluMist® CA07
(H1N1pdm) was plaque purified in MDCK cells from FluMist® 2011-2012 vaccine Lot#
B11K1802. The A/California/07/2009 (CA07) HA (SEQ ID NO: 26) and NA (SEQ ID NO: 27)
sequences used are shown below.
98
48204255.2
A/California/07/2009 (H1N1) HA in M2KOTMdel
AGCAAAAGCAGGGGAAAACAAAAGCAACAAAAATGAAGGCAATACTAGTAGTTCTGCTATATACATTTGCAACCGCAAAT
GCAGACACATTATGTATAGGTTATCATGCGAACAATTCAACAGACACTGTAGACACAGTACTAGAAAAGAATGTAACAGT
AACACACTCTGTTAACCTTCTAGAAGACAAGCATAACGGGAAACTATGCAAACTAAGAGGGGTAGCCCCATTGCATTTGG
GTAAATGTAACATTGCTGGCTGGATCCTGGGAAATCCAGAGTGTGAATCACTCTCCACAGCAAGCTCATGGTCCTACATT
GTGGAAACACCTAGTTCAGACAATGGAACGTGTTACCCAGGAGATTTCATCGATTATGAGGAGCTAAGAGAGCAATTGAG
CTCAGTGTCATCATTTGAAAGGTTTGAGATATTCCCCAAGACAAGTTCATGGCCCAATCATGACTCGAACAAAGGTGTAA
CGGCAGCATGTCCTCATGCTGGAGCAAAAAGCTTCTACAAAAATTTAATATGGCTAGTTAAAAAAGGAAATTCATACCCA
AAGCTCAGCAAATCCTACATTAATGATAAAGGGAAAGAAGTCCTCGTGCTATGGGGCATTCACCATCCATCTACTAGTGC
TGACCAACAAAGTCTCTATCAGAATGCAGATGCATATGTTTTTGTGGGGTCATCAAGATACAGCAAGAMGTTCAAGCCGG
AAATAGCAATAAGACCCAAAGTGAGGGATCRAGAAGGGAGAATGAACTATTACTGGACACTAGTAGAGCCGGGAGACAAA
ATAACATTCGAAGCAACTGGAAATCTAGTGGTACCGAGATATGCATTCGCAATGGAAAGAAATGCTGGATCTGGTATTAT
CATTTCAGATACACCAGTCCACGATTGCAATACAACTTGTCAAACACCCAAGGGTGCTATAAACACCAGCCTCCCATTTC
AGAATATACATCCGATCACAATTGGAAAATGTCCAAAATATGTAAAAAGCACAAAATTGAGACTGGCCACAGGATTGAGG
AATATCCCGTCTATTCAATCTAGAGGCCTATTTGGGGCCATTGCCGGTTTCATTGAAGGGGGGTGGACAGGGATGGTAGA
TGGATGGTACGGTTATCACCATCAAAATGAGCAGGGGTCAGGATATGCAGCCGACCTGAAGAGCACACAGAATGCCATTG
ACGAGATTACTAACAAAGTAAATTCTGTTATTGAAAAGATGAATACACAGTTCACAGCAGTAGGTAAAGAGTTCAACCAC
CTGGAAAAAAGAATAGAGAATTTAAATAAAAAAGTTGATGATGGTTTCCTGGACATTTGGACTTACAATGCCGAACTGTT
GGTTCTATTGGAAAATGAAAGAACTTTGGACTACCACGATTCAAATGTGAAGAACTTATATGAAAAGGTAAGAAGCCAGC
TAAAAAACAATGCCAAGGAAATTGGAAACGGCTGCTTTGAATTTTACCACAAATGCGATAACACGTGCATGGAAAGTGTC
AAAAATGGGACTTATGACTACCCAAAATACTCAGAGGAAGCAAAATTAAACAGAGAAGAAATAGATGGGGTAAAGCTGGA
ATCAACAAGGATTTACCAGATTTTGGCGATCTATTCAACTGTCGCCAGTTCATTGGTACTGGTAGTCTCCCTGGGGGCAA
TCAGTTTCTGGATGTGCTCTAATGGGTCTCTACAGTGTAGAATATGTATTTAACATTAGGATTTCAGAAGCATGAGAAAA
AAACACCCTTGTTTCTACT
> A/California/07/2009 (H1N1) NA in M2KOTMdel
AGCAAAAGCAGGAGTTTAAAATGAATCCAAACCAAAAGATAATAACCATTGGTTCGGTCTGTATGACAATTGGAATGGCT
AACTTAATATTACAAATTGGAAACATAATCTCAATATGGATTAGCCACTCAATTCAACTTGGGAATCAAAATCAGATTGA
AACATGCAATCAAAGCGTCATTACTTATGAAAACAACACTTGGGTAAATCAGACATATGTTAACATCAGCAACACCAACT
TTGCTGCTGGACAGTCAGTGGTTTCCGTGAAATTAGCGGGCAATTCCTCTCTCTGCCCTGTTAGTGGATGGGCTATATAC
AGTAAAGACAACAGTGTAAGAATCGGTTCCAAGGGGGATGTGTTTGTCATAAGGGAACCATTCATATCATGCTCCCCCTT
GGAATGCAGAACCTTCTTCTTGACTCAAGGGGCCTTGCTAAATGACAAACATTCCAATGGAACCATTAAAGACAGGAGCC
CATATCGAACCCTAATGAGCTGTCCTATTGGTGAAGTTCCCTCTCCATACAACTCAAGATTTGAGTCAGTCGCTTGGTCA
GCAAGTGCTTGTCATGATGGCATCAATTGGCTAACAATTGGAATTTCTGGCCCAGACAATGGGGCAGTGGCTGTGTTAAA
GTACAACGGCATAATAACAGACACTATCAAGAGTTGGAGAAACAATATATTGAGAACACAAGAGTCTGAATGTGCATGTG
TAAATGGTTCTTGCTTTACTGTAATGACCGATGGACCAAGTAATGGACAGGCCTCATACAAGATCTTCAGAATAGAAAAG
GGAAAGATAGTCAAATCAGTCGAAATGAATGCCCCTAATTATCACTATGAGGAATGCTCCTGTTATCCTGATTCTAGTGA
AATCACATGTGTGTGCAGGGATAACTGGCATGGCTCGAATCGACCGTGGGTGTCTTTCAACCAGAATCTGGAATATCAGA
TAGGATACATATGCAGTGGGATTTTCGGAGACAATCCACGCCCTAATGATAAGACAGGCAGTTGTGGTCCAGTATCGTCT
AATGGAGCAAATGGAGTAAAAGGGTTTTCATTCAAATACGGCAATGGTGTTTGGATAGGGAGAACTAAAAGCATTAGTTC
AAGAAACGGTTTTGAGATGATTTGGGATCCGAACGGATGGACTGGGACAGACAATAACTTCTCAATAAAGCAAGATATCG
TAGGAATAAATGAGTGGTCAGGATATAGCGGGAGTTTTGTTCAGCATCCAGAACTAACAGGGCTGGATTGTATAAGACCT
TGCTTCTGGGTTGAACTAATCAGAGGGCGACCCAAAGAGAACACAATCTGGACTAGCGGGAGCAGCATATCCTTTTGTGG
TGTAAACAGTGACACTGTGGGTTGGTCTTGGCCAGACGGTGCTGAGTTGCCATTTACCATTGACAAGTAATTTGTTCAAA
AAACTCCTTGTTTCTACT
99
48204255.2
[0305] Mice (7-8 weeks old, female BALB/c) were intranasally inoculated with M2KO(ΔTM)
CA07 (106 TCID50/mouse), M2WT CA07 (106 TCID50/mouse), FluMist® CA07 (106
TCID50/mouse) or OPTI-MEM™ as naïve control. Body weight and clinical symptoms were
observed for 14 days post-inoculation. Figure 37 shows that M2KO(ΔTM) and FluMist®
vaccinated mice did not lose weight whereas the virus that contains the WT M2 loses weight and
succumbs to infection. These results demonstrate that deletion of the M2 gene attenuates the
virus and that M2KO(ΔTM) is attenuated.
[0306] Figure 38 M2KO(ΔTM), FluMist,® and M2 wild-type viral titers lungs and nasal
terminates. Lungs and nasal turbinates were harvested on day 3 post-vaccination for titration of
virus on cells. No virus was detected in either the lungs or the nasal turbinates in the
M2KO(ΔTM) immunized mice. In contrast, FluMist® did have virus replication in both the
lung and nasal turbinates although at lower levels than the wild-type virus.
[0307] Figure 39 M2KO(ΔTM) and FluMist® titers in sera collected 7, 14, and 21 days postinoculation and anti-HA IgG titers were determined by ELISA. M2KO(ΔTM) induced higher
responses that were detected earlier than FluMist® responses. By day 21 peak antibody levels
were reached by both viruses.
[0308] Figure 40 shows the percent survival of mice challenged 12 weeks post-immunization
with 40 MLD50 of heterologous virus, mouse-adapted influenza A/Aichi/2/1968 (H3N2). Body
weight change and clinical symptoms were observed for 14 days after challenge. All the
M2KO(ΔTM) (H1N1pdm HA ,NA) immunized mice were protected against the Aichi (H3N2)
challenge whereas only 80% of the FluMist® (H1N1pdm HA ,NA) were protected. The
surviving FluMist® mice lost close to 20% of their body weight whereas M2KO(ΔTM) mice lost
~10% of their body weight.
[0309] Table 26 shows the virus titers in the lungs and nasal turbinates that were collected on
day 3 post-challenge. M2KO(ΔTM) and FluMist® controlled challenge virus replication in the
100
48204255.2
lungs and nasal turbinates to similar levels whereas naïve mice displayed virus titers that were a
log higher in both the lung and the nasal turbinates.
[0310] Intracellular staining of cells in bronchoalveolar lavage (BAL). BAL was collected 3
days post-challenge and stained with surface markers for immunostaining by flow cytometry to
detect CD8+CD4+, CD8+CD4-, CD8-CD4+, CD8-CD4- cell populations. Both CD4+ and
CD8+ cell populations were greater in the vaccinated mice than the naïve mice indicating that
M2KO(ΔTM) primed for a cellular response similar to FluMist®.. M2KO(ΔTM) vaccinated
mice had greater CD8+CD4- cell population than FluMist® (49% vs 40%) (Figure 41).
Table 26. Virus titers in respiratory tract of mice.
Example 19 M2KO(ΔTM) mRNA Expression Relative to FluMist® and Wild-Type Virus
[0311] In some embodiments, the M2KO(ΔTM) virus is produced in cells that stably provide
M2 protein in trans resulting in a virus that has functional M2 protein in the viral membrane but
does not encode M2 in its genome. Therefore, we hypothesize that the M2KO(ΔTM) virus
behaves similar to wild-type virus in the initial infection and first round of replication in normal
cells. We suggest that mRNA levels of viral antigens are similar to wild-type levels early in
infection and stimulate a potent immune response sooner than attenuated replicating viral
vaccines.
Lung
(log pfu/g)
Nasal Turbinate
(log pfu/g)
M2KO CA07 5.95 ± 0.59 5.61 ± 0.47
FluMist CA07 5.94 ± 0.46 3.88 ± 0.64
Naive 6.86 ± 0.06 6.52 ± 1.05
101
48204255.2
[0312] Human lung carcinoma (A549) cells were infected at a multiplicity of infection of 0.5
with M2KO(ΔTM), FluMist® and wild-type viruses. Unadsorbed virus was removed by washing
five times with PBS. After addition of virus growth media, the infected cells were placed in the
35oC CO2 incubator. No trypsin was added to the growth medium to ensure single-cycle
replication for all viruses. Cell monolayers were harvested and RNA extracted at 4, 9 and 22
hours post infection.
[0313] Total RNA (100ng) from control and infected A549 cells were used for quantitative
RT-PCR analysis. cDNA was synthesized with oligo-dT primers and Superscript II reverse
transcriptase(Invitrogen) and quantified by real-time quantitative PCR analysis using genespecific primers for an early influenza gene, M1, and a late influenza gene, HA and cytokine IP10 gene. Reactions were performed using SYBR Green reagent (Invitrogen, Carlsbad) according
to the manufacturer’s instructions. Reaction efficiency was calculated by using serial 10-fold
dilutions of the housekeeping gene ɣ-actin and sample genes. Reactions were carried out on an
ABI 7300 realtime PCR system (Applied Biosystems, Foster City, CA,USA) and the thermal
profile used was Stage 1: 50°C for 30 min; Stage 2: 95°C for 15 min; Stage 3: 94°C for 15 sec,
55°C for 30 sec; and 72°C for 30 sec, repeated for 30 cycles. All quantitations (threshold cycle
[CT] values) were normalized to that of the housekeeping gene to generate ΔCT, and the
difference among the ΔCT value of the sample and that of the reference (wild-type sample) was
calculated as -ΔΔCT. The relative level of mRNA expression was expressed as 2-ΔΔCT.
[0314] M2KO(ΔTM) virus HA mRNA expression was similar to wild-type M2 virus for H3
(Table 27), PR8 (Table 28) and H1N1pdm (Figure 42) at 4 hour post-infection. Cold-adapted
FluMist® was less than wild-type and M2KO(ΔTM) in the early timepoints due to slower
replication kinetics. When M1, an early timepoint gene, mRNA expression was tested, similar
results were observed (Table 27, Figure 42). These results suggest that M2KO(ΔTM) generates
similar levels of mRNA in the early infection cycle to produce de novo viral antigens that create
a ‘danger signal’ similar to wild-type virus and induce a potent immune response.
102
48204255.2
Table 27. Relative mRNA expression of H3 HA genes.
Table 28. Relative mRNA expression of the PR8 HA and M1 genes.
Example 20: Generation of M2 Vero Production Cells
[0315] The M2 gene of PR8 virus was cloned into expression vector pCMV-SC (Stratagene,
La Jolla, CA) by standard molecular techniques to generate pCMV-PR8-M2. The plasmid was
digested with EcoR1 to confirm the presence of the 300 bp M2 gene and the 4.5 Kb vector as
shown in Figure 43. The sequence of the plasmid containing the M2 gene insert was confirmed
as shown in Figure 44.
Sample neat 1:10 neat 1:10
4 hr p.i. PR8 WT 1.00 1.00 1.00 1.00
PR8 M2KOTMdel 3.07 1.29 3.15 2.64
Mock 0.02 0.30 0.15 0.32
9 hr p.i. PR8 WT 1.00 1.00 1.00 1.00
PR8 M2KOTMdel 2.05 3.27 4.04 5.11
Mock 0.00 0.00 0.00 0.00
HA M1
103
48204255.2
[0316] Generation of M2 Vero cells: The pCMV-PR8-M2 plasmid described earlier and
containing a neomycin resistant gene, was transfected into Vero cells (ATCC CCL-81) by using
the Trans IT-LT1 transfection reagent (Mirus) according to the manufacturer’s instructions.
Briefly, on the day before transfection, Vero cells were plated at 5x105 cells/100-mm dish. On
day 1, 10 µg of plasmid DNA was mixed with 20 µg of Trans IT-LT1 in 0.3 ml of OptiMEM
(Invitrogen) and was incubated with these cells at 37°C in 5% CO2 overnight. On day 2, the
transfection mixture was replaced with a complete medium that is modified Eagle’s medium
(MEM) supplemented with 5% newborn calf serum. The medium also contained 1mg/ml of
geneticin (Invitrogen), a broad spectrum antibiotic that is used to select mammalian cells
expressing the neomycin protein. Resistant cells (Vero cells stably expressing M2 gene) began
to grow in the selection medium, the medium was replaced with fresh selection medium and
geneticin-resistant clones were isolated by limited dilution in TC-96 plates. The surface
expression of the M2 protein was demonstrated by immunostaining using a M2 specific
monoclonal antibody, 14C2 (Santa Cruz Biotechnology).
[0317] Infection of parental and modified M2 Vero cells with M2KO(ΔTM) virus: The ability
of M2 Vero cells to serve as production cells for M2KO(ΔTM) virus was tested by infection with
M2KO(ΔTM)-PR8 virus. Briefly, monolayers of M2 Vero and parent Vero cells were infected
with ten-fold serial dilutions (10-1 to 10-6) of M2KO(ΔTM)-PR8 virus using standard influenza
infection procedures. The infected cells were incubated at 35oC and observed for cytopathic
effect (CPE) daily. Only M2 Vero cells displayed CPE indicating virus growth. Supernatant
was harvested on day 4 from the 10-3 well and virus titer was determined by TCID50 assay on
MDCK cells stably expressing M2 gene (M2CK). M2KO(ΔTM)-PR8 virus titer grown in M2
Vero cells was 106.75 TCID50/ml indicating that M2 Vero cells can serve as production cells for
the manufacture of M2KO(ΔTM) vaccine.
104
48204255.2
Example 21: Intradermal Delivery of Influenza Vaccines
[0318] This example demonstrates the immunogenicity of the seasonal influenza vaccine,
FluLaval (2011-2012 formulation), when administered intramuscularly (IM), intradermally (ID),
and using a subcutaneous microneedle device such as that described in published U.S. Patent
Application 2011/0172609. Hairless guinea pigs were inoculated on day 0 and select groups
were boosted on day 30. Sera was collected on days 0, 30 and 60 and analyzed by enzymelinked immunosorbent assay (ELISA) for hemagglutinin-specific IgG responses.
[0314] Results are shown in Figures 51-53. The data shows qualitative absorbance of antibody
levels to the three strains formulated in the seasonal influenza vaccine FluLaval:
A/California/7/2009 NYMC X-181, A/Victoria/210/2009 NYMC X-187 (an A/Perth/16/2009-
like virus), and B/Brisbane/60/2008. At day 30, IM and ID delivery produced identical IgG
responses to all viral HA. The ID prime only groups displayed higher titers at day 60, suggesting
that ID delivery induces long lasting immunity to all viral HA.
[0315] The term “comprising” as used in this specification and claims means “consisting at
least in part of”. When interpreting statements in this specification and claims which include
“comprising”, other features besides the features prefaced by this term in each statement can also
be present. Related terms such as “comprise” and “comprised” are to be interpreted in a similar
manner.
[0316] In this specification where reference has been made to patent specifications, other
external documents, or other sources of information, this is generally for the purpose of
providing a context for discussing the features of the invention. Unless specifically stated
otherwise, reference to such external documents is not to be construed as an admission that such
documents, or such sources of information, in any jurisdiction, are prior art, or form part of the
common general knowledge in the art.
Claims (24)
1. A recombinant influenza virus having a mutant M gene comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
2. The recombinant influenza virus of claim 1, wherein mutation of the M gene results in failure of the virus to express the M2 protein, or causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:4.
3. The recombinant influenza virus of claim 1, wherein the mutant M gene does not revert to wild-type or to a non-wild-type sequence encoding a functional M2 protein for at least 10 passages in an in vitro host cell system, wherein the host cell is modified to produce a wild-type version of the mutant gene, thereby providing the gene product to the virus in trans.
4. The recombinant virus of claim 1, wherein the virus is an influenza A virus.
5 The recombinant virus of claim 1, wherein the virus is non-pathogenic in a mammal infected with the virus.
6. The recombinant virus of claim 3, wherein the in vitro cell system comprises Chinese Hamster Ovary cells or Vero cells.
7. A composition comprising: a recombinant influenza virus having a mutant M gene comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
8. The composition of claim 7, wherein mutation of the M gene results in failure of the virus to express the M2 protein, or causes the virus to express a truncated M2 protein having the amino acid sequence of SEQ ID NO:4.
9. The composition of claim 7, wherein the virus is an influenza A virus. 106 48204255.2
10. The composition of claim 7, wherein the composition is non-pathogenic to a mammal administered the composition.
11. The composition of claim 7, wherein the composition elicits a detectable immune response in a mammal within about three weeks after administration of the composition to the mammal.
12. A method for propagating a recombinant influenza virus, comprising: contacting an isolated host cell with a recombinant influenza virus comprising SEQ ID NO:1, SEQ ID NO:2,or SEQ ID NO: 3; and incubating the host cell for a sufficient time and under conditions suitable for viral replication, wherein the host cell is modified to produce a wild-type version of the influenza M gene, thereby providing the gene product to the virus in trans.
13. The method of claim 12, further comprising isolating progeny virus particles.
14. The method of claim 13, further comprising formulating the virus particles into a vaccine.
15. The method of claim 12, wherein the virus fails to express the M2 protein, or expresses a truncated M2 protein having the amino acid sequence of SEQ ID NO:4.
16. The method of claim 12, wherein the virus is an influenza A virus.
17. The method of claim 12, wherein the virus is non-pathogenic to a mammal administered the virus.
18. The method of claim 12, wherein the virus elicits a detectable immune response in a mammal within about three weeks after administration of a composition comprising the virus to the mammal. 107 48204255.2
19. The method of claim 12, wherein the mutant M gene does not revert to wild-type or to a non-wild-type sequence encoding a functional M2 protein for at least 10 passages of the host cell.
20. The method of claim 12, wherein the host cell is a CHO cell or a Vero cell.
21. A recombinant influenza virus of any one of claims 1-6 substantially as herein described with reference to any example thereof and with or without reference to the accompanying figures.
22. A composition of any one of claims 7-11 substantially as herein described with reference to any example thereof and with or without reference to the accompanying figures.
23. A method of any one of claims 12-20 substantially as herein described with reference to any example thereof and with or without reference to the accompanying figures.
24. A recombinant influenza virus when propagated by a method of any one of claims 12-20 and 23.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161501034P | 2011-06-24 | 2011-06-24 | |
US61/501,034 | 2011-06-24 | ||
PCT/US2012/043606 WO2012177924A2 (en) | 2011-06-24 | 2012-06-21 | Influenza virus mutants and uses therefor |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ620242A NZ620242A (en) | 2016-02-26 |
NZ620242B2 true NZ620242B2 (en) | 2016-05-27 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11980661B2 (en) | Influenza virus mutants and uses therefor | |
EP3119883A2 (en) | Influenza virus vectors and uses therefor | |
NZ620242B2 (en) | Influenza virus mutants and uses therefor |